Conserved HBV and HCV sequences useful for gene silencing

ABSTRACT

Conserved consensus sequences from known hepatitis B virus strains and known hepatitis C virus strains, which are useful in inhibiting the expression of the viruses in mammalian cells, are provided. These sequences are useful to silence the genes of HBV and HCV, thereby providing therapeutic utility against HBV and HCV viral infection in humans.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation under 35 USC § 120 of U.S. patentapplication Ser. No. 14/921,510 filed Oct. 23, 2015, which is acontinuation under 35 USC § 120 of U.S. patent application Ser. No.14/043,272 filed Oct. 1, 2013, now U.S. Pat. No. 9,200,281, issued onDec. 1, 2015, which continuation under 35 USC § 120 of U.S. patentapplication Ser. No. 13/065,601 filed Dec. 12, 2005, now U.S. Pat. No.8,575,327, issued on Nov. 5, 2013, which is a continuation-in-part under35 USC § 120 of International Application PCT/US2004/019229 filed Jun.10, 2004, which claims benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application 60/478,076 filed Jun. 12, 2003, the contents ofeach of which are incorporated herein by reference in their entireties.U.S. patent application Ser. No. 13/065,601 also claims benefit under 35U.S.C. § 119(e) of U.S. Provisional Application No. 60/638,294, filedDec. 22, 2004, which is incorporated herein by reference in itsentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Apr. 27, 2018, isnamed 051058-034400-C3_SL.txt and is 63,829 bytes in size.

FIELD OF THE INVENTION

This invention relates to methods and compositions utilizing conservedgenetic sequences of known hepatitis B viral (HBV) strains and knownhepatitis C viral (HCV) strains to modulate the expression of HBV and/orHCV in mammalian cells, via double-stranded RNA-mediated gene silencing,including post-transcriptional gene silencing (PTGS) and transcriptionalgene silencing (TGS).

BACKGROUND OF THE INVENTION

Human hepatitis C (HCV) is a major public health problem with anestimated 200 million persons worldwide infected. The number of newinfections per year in the United States is estimated to be about 25,000in 2001. This number has declined from an estimated 240,000 new casesper year in the 1980's due to blood donor screening. Nevertheless, anestimated 3.9 million (1.8%) Americans have been infected with HCV, ofwhom 2.7 million are chronically infected. Hepatitis C shows significantgenetic variation in worldwide populations, evidence of its frequentrates of mutation and rapid evolution. There are six basic genotypes ofHCV, with 15 recorded subtypes, which vary in prevalence acrossdifferent regions of the world. Each of these major genotypes may differsignificantly in their biological effects—in terms of replication,mutation rates, type and severity of liver damage, and detection andtreatment options—however, these differences are not yet clearlyunderstood.

There is currently no vaccine against HCV and available drug therapy,including ribavirin and interferon, is only partially effective. It isestimated that 75-85% of infected persons will develop a chronicinfection, with 70% of chronically infected persons expected to developchronic liver 5 disease including hepatocellular carcinoma. Chronic HCVrelated liver disease is a leading indication for liver transplant.

Although a human hepatitis B vaccine has been available since 1982, itis estimated that 350 million persons worldwide are chronically infectedwith HBV. While the number of new infections per year in the UnitedStates has declined from an average of 260,000 in the 1980s to about78,000 in 2001, there are an estimated 1.25 million hepatitis Bcarriers, defined as persons positive for hepatitis B surface antigen(HBsAg) for more than 6 months. Such carriers of HBV are at increasedrisk for developing cirrhosis, hepatic decompensation, andhepatocellular carcinoma. Although most carriers do not develop hepaticcomplications from chronic hepatitis B, 15% to 40% will develop serioussequelae during their lifetime, and death from chronic liver diseaseoccurs in 15-25% of chronically infected persons.

There is a need for improved therapeutic agents effective in patientssuffering from HBV and/or HCV infection, especially chronic infection,which together are estimated to account for 75% of all cases of liverdisease around the world. There is also an extreme need for prophylacticmethods and agents effective against HCV.

Nucleic acids (e.g., DNA, RNA, hybrid, heteroduplex, and modifiednucleic acids) have come to be recognized as extremely valuable agentswith significant and varied biological activities, including their useas therapeutic moieties in the prevention and/or treatment of diseasestates in man and animals. For example, oligonucleotides acting throughantisense mechanisms are designed to hybridize to target mRNAs, therebymodulating the activity of the mRNA. Another approach to the utilizationof nucleic acids as therapeutics is designed to take advantage oftriplex or triple strand formation, in which a single-stranded oligomer(e.g., DNA or RNA) is designed to bind to a double-stranded DNA targetto produce a desired result, e.g., inhibition of transcription from theDNA target. Yet another approach to the utilization of nucleic acids astherapeutics is designed to take advantage of ribozymes, in which astructured RNA or a modified oligomer is designed to bind to an RNA or adouble-stranded DNA target to produce a desired result, e.g., targetedcleavage of RNA or the DNA target and thus inhibiting its expression.Nucleic acids may also be used as immunizing agents, e.g., byintroducing DNA molecules into the tissues or cells of an organism thatexpress proteins capable of eliciting an immune response. Nucleic acidsmay also be engineered to encode an RNA with antisense, ribozyme, ortriplex activities, or to produce RNA that is translated to produceprotein(s) that have biological function.

More recently, the phenomenon of RNAi or double-stranded RNA(dsRNA)-mediated gene silencing has been recognized, whereby dsRNAcomplementary to a region of a target gene in a cell or organisminhibits expression of the target gene (see, e.g., WO 99/32619,published 1 Jul. 1999, Fire et al.; and U.S. Pat. No. 6,506,559:“Genetic Inhibition by Double-Stranded RNA;” WO 00/63364: “Methods andCompositions for Inhibiting the Function of Polynucleotide Sequences,”Pachuk and Satishchandran; and U.S. Ser. No. 60/419,532, filed Oct. 18,2002). dsRNA-mediated gene silencing, utilizing compositions providingan at least partially double-stranded dsRNA, is expected to provideextremely valuable therapeutic and/or prophylactic agents against viralinfection, including HBV and/or HCV, including in the extremelydifficult problem of chronic HBV and/or HCV infection.

SUMMARY OF THE INVENTION

A method for inhibiting expression of a polynucleotide sequence ofhepatitis B virus in an in vivo mammalian cell comprising administeringto said cell at least one double-stranded RNA effector molecule,preferably 2, 3, 4, 5, 6, or more double-stranded RNA effectormolecules, each double-stranded RNA effector molecule comprising asequence selected from the group consisting of SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:49;wherein U is substituted for T. In a preferred method, three or fourdsRNA effector molecules, each comprising a sequence selected from thegroup consisting of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:23, and SEQ IDNO:49; wherein U is substituted for T; are administered to an in vivomammalian cell. The double-stranded RNA effector molecules may beprepared exogenously and administered into a mammalian cell or expressedintracellularly in a mammalian cell from a double-stranded RNAexpression vector, i.e., an expression vector engineered to express adsRNA effector molecule in a mammalian cell. In a preferred method, atleast three or four dsRNA effector molecules, each comprising a sequenceselected from the group consisting of SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:23, and SEQ ID NO:49; wherein U is substituted for T; are encoded ina dsRNA expression vector which is administered to an in vivo mammaliancell.

A composition for inhibiting the expression of a polynucleotide sequenceof hepatitis B virus in an in vivo mammalian cell comprising at leastone, preferably 2, 3, 4, 5, 6 or more double-stranded RNA effectormolecules, each double-stranded RNA effector molecule comprising asequence selected from the group consisting of SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:49;wherein U is substituted for T. In a preferred composition, at leastthree or four dsRNA effector molecules are included, each comprising asequence selected from the group consisting of SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:23, and SEQ ID NO:49; wherein U is substituted for T.The double-stranded RNA effector molecules may be preparedexogenogenously and the composition comprising two, three, four, five,six, or more dsRNA effector molecules administered into a mammaliancell, or the composition may comprise one or more dsRNA expressionconstructs capable of expressing in a mammalian cell two, three, four,five, six or more of said dsRNA effector molecules. In a preferredcomposition, three or four dsRNA effector molecules, each comprising asequence selected from the group consisting of SEQ ID NO:18, SEQ IDNO:19, SEQ ID NO:23, and SEQ ID NO:49; wherein U is substituted for T,are encoded in a dsRNA expression vector.

A method for inhibiting expression of a polynucleotide sequence ofhepatitis B virus in an in vivo mammalian cell comprising administeringto said cell at least two, preferably 3, 4, 5, 6 or more,double-stranded RNA effector molecules, each double-stranded RNAeffector molecule comprising: (a) a sequence selected from the groupconsisting of SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO:57, SEQ ID NO:58,SEQ ID NO:59, and SEQ ID NO:62; (b) the reverse complement of saidselected sequence; and (c) optionally, a sequence linking sequences (a)and (b); wherein U is substituted for T. In a preferred method, saiddsRNA effector molecules will comprise 3 or 4 sequences selected fromthe group consisting of SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO:59, andSEQ ID NO:62; wherein U is substituted for T. The double-stranded RNAeffector molecules may be stem-loop or hairpin structures and/or duplexdouble-stranded RNA molecules. The double-stranded RNA effectormolecules may be prepared exogenogenously and the two, three, four,five, six, or more dsRNA effector molecules administered into amammalian cell, or one or more dsRNA expression constructs capable ofexpressing in a mammalian cell two, three, four, five, six or more ofsaid dsRNA effector molecules may be administered.

A composition for inhibiting expression of a polynucleotide sequence ofhepatitis B virus in an in vivo mammalian cell comprising at least twodouble-stranded RNA effector molecules, each double-stranded RNAeffector molecule comprising: (a) a sequence selected from the groupconsisting of SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO:57, SEQ ID NO:58,SEQ ID NO:59, and SEQ ID NO:62; (b) the reverse complement of saidselected sequence; and (c) optionally, a sequence linking sequences (a)and (b); wherein U is substituted for T. In a preferred composition,three or four of said dsRNA effector molecules will be included, orencoded in an expression vector, comprising sequences selected from thegroup consisting of SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO:59, and SEQID NO:62; wherein U is substituted for T. The double-stranded RNAeffector molecules may be prepared exogenously and the composition willcomprise two, three, four, five, six, or more of said dsRNA effectormolecules for administration into a in vivo mammalian cell, or thecomposition may comprise one or more dsRNA expression constructs capableof expressing in a mammalian cell two, three, four, five, six or more ofsaid dsRNA effector molecules.

In another aspect the invention relates to methods and compositions forinhibiting expression of a polynucleotide sequence of hepatitis B virusin an in vivo mammalian cell comprising administering to said cell atleast two, preferably 3, 4, 5, 6 or more, double-stranded RNA effectormolecules, each double-stranded RNA effector molecule comprising: (a) asequence selected from the group consisting of SEQ ID NO: 50; SEQ ID NO:51; SEQ ID NO: 52; SEQ ID NO:53; SEQ ID NO: 54, SEQ ID NO: 55, SEQ IDNO: 56; SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO: 60; SEQ IDNO:61; and SEQ ID NO:62; (b) the reverse complement of said selectedsequence; and (c) optionally, a sequence linking sequences (a) and (b);wherein U is substituted for T.

A polynucleotide sequence comprising SEQ ID NO:49.

A method for inhibiting expression of a polynucleotide sequence ofhepatitis C virus in an in vivo mammalian cell comprising administeringto said cell at least one double-stranded RNA effector molecule,preferably 2, 3, 4, 5, 6, or more double-stranded RNA effectormolecules, comprising (a) an RNA sequence equivalent to a hepatitis Cvirus DNA coding strand sequence selected from the group consisting ofsequence position 9510-9531, 9510-9533, 9510-9534, 9510-9535, 9510-9536,9514-9534, 9514-9535, 9514-9536, 9514-9539, 9514-9540, 9514-9542,9517-9539, 9517-9540, 9517-9542, 9517-9544, 9518-9539, 9518-9540,9518-9542, 9518-9544, 9520-9540, 9520-9542, 9520-9544, 9520-9548,9521-9542, 9521-9544, 9521-9548, 9521-9549, 9522-9542, 9522-9544,9522-9548, 9522-9549, 9527-9548, 9527-9549, 9527-9551, 9527-9552,9527-9553, 9527-9555, 9528-9548, 9528-9549, 9528-9551, 9528-9552,9528-9553, 9528-9555, 9530-9551, 9530-9552, 9530-9553, 9530-9555,9530-9557, 9530-9558, 9532-9552, 9532-9553, 9532-9555, 9532-9557,9532-9558, 9532-9559, 9532-9560, 9537-9557, 9537-9558, 9537-9559,9537-9560, 9537-9561, 9537-9564, 9538-9558, 9538-9559, 9538-9560,9538-9561, 9538-9564, 9538-9566, 9541-9561, 9541-9564, 9541-9566,9541-9568, 9541-9569, 9543-9564, 9543-9566, 9543-9568, 9543-9569,9543-9571, 9545-9566, 9545-9568, 9545-9569, 9545-9571, 9545-9573,9546-9564, 9546-9566, 9546-9569, 9546-9571, 9546-9573, 9547-9568,9547-9569, 9547-9571, 9547-9573, 9547-9575, 9550-9571, 9550-9573,9550-9575, 9550-9577, 9550-9578, 9554-9575, 9554-9577, 9554-9578,9554-9580, 9556-9577, 9556-9578, 9556-9580, 9556-9584, 9562-9584,9562-9586, 9562-9587, 9562-9588, 9562-9589, 9563-9584, 9563-9586,9563-9587, 9563-9588, 9563-9589, 9563-9591, 9565-9586, 9565-9587,9565-9588, 9565-9589, 9565-9591, 9565-9593, 9567-9587, 9567-9588,9567-9589, 9567-9591, 9567-9593, 9567-9595, 9570-9591, 9570-9593,9570-9595, 9570-9596, 9570-9598, 9572-9593, 9572-9595, 9572-9596,9572-9598, 9574-9595, 9574-9596, 9574-9598, 9574-9601, 9576-9596,9576-9598, 9576-9601, 9576-9604, 9579-9601, 9579-9604, 9581-9601,9581-9604, and 9583-9604 and (b) an RNA sequence which is the reversecomplement of the selected sequence equivalent to the hepatitis C virusDNA coding strand sequence. In some embodiments, said RNA sequences (a)and (b) are linked by a loop sequence and the double-stranded RNAeffector molecule(s) forms a stem-loop or hairpin dsRNA structure. Insome aspects, said double-stranded RNA effector molecule(s) are duplexdsRNAs, formed from two separate RNA strands. In some aspects, themethod involves administering to a mammalian cell an expressionconstruct encoding one, two, three, four, five or more of said dsRNAeffector molecules. In some embodiments designed to target the HCV minusstrand, the dsRNA effector molecule will comprise (a) an RNA sequencecorresponding to a hepatitis C virus DNA coding strand sequence asspecified above, and (b) the reverse complement of said RNA sequence,optionally linked by a loop sequence. In some embodiments, the dsRNAeffector molecule(s) is encoded by an expression construct.

In some aspects the invention relates to a composition for inhibitingthe expression of a polynucleotide sequence of hepatitis C virus in anin vivo mammalian cell comprising at least one double-stranded RNAeffector molecule, preferably 2, 3, 4, 5, 6 or more double-stranded RNAeffector molecules, or a dsRNA expression construct capable oftranscribing one, 2, 3, 4, 5, 6 or more of said dsRNA effector moleculesin an in vivo mammalian cell, each of said dsRNA effector moleculescomprising (a) an RNA sequence equivalent to a hepatitis C virus DNAcoding strand sequence selected from the group consisting of sequenceposition 9510-9531, 9510-9533, 9510-9534, 9510-9535, 9510-9536,9514-9534, 9514-9535, 9514-9536, 9514-9539, 9514-9540, 9514-9542,9517-9539, 9517-9540, 9517-9542, 9517-9544, 9518-9539, 9518-9540,9518-9542, 9518-9544, 9520-9540, 9520-9542, 9520-9544, 9520-9548,9521-9542, 9521-9544, 9521-9548, 9521-9549, 9522-9542, 9522-9544,9522-9548, 9522-9549, 9527-9548, 9527-9549, 9527-9551, 9527-9552,9527-9553, 9527-9555, 9528-9548, 9528-9549, 9528-9551, 9528-9552,9528-9553, 9528-9555, 9530-9551, 9530-9552, 9530-9553, 9530-9555,9530-9557, 9530-9558, 9532-9552, 9532-9553, 9532-9555, 9532-9557,9532-9558, 9532-9559, 9532-9560, 9537-9557, 9537-9558, 9537-9559,9537-9560, 9537-9561, 9537-9564, 9538-9558, 9538-9559, 9538-9560,9538-9561, 9538-9564, 9538-9566, 9541-9561, 9541-9564, 9541-9566,9541-9568, 9541-9569, 9543-9564, 9543-9566, 9543-9568, 9543-9569,9543-9571, 9545-9566, 9545-9568, 9545-9569, 9545-9571, 9545-9573,9546-9564, 9546-9566, 9546-9569, 9546-9571, 9546-9573, 9547-9568,9547-9569, 9547-9571, 9547-9573, 9547-9575, 9550-9571, 9550-9573,9550-9575, 9550-9577, 9550-9578, 9554-9575, 9554-9577, 9554-9578,9554-9580, 9556-9577, 9556-9578, 9556-9580, 9556-9584, 9562-9584,9562-9586, 9562-9587, 9562-9588, 9562-9589, 9563-9584, 9563-9586,9563-9587, 9563-9588, 9563-9589, 9563-9591, 9565-9586, 9565-9587,9565-9588, 9565-9589, 9565-9591, 9565-9593, 9567-9587, 9567-9588,9567-9589, 9567-9591, 9567-9593, 9567-9595, 9570-9591, 9570-9593,9570-9595, 9570-9596, 9570-9598, 9572-9593, 9572-9595, 9572-9596,9572-9598, 9574-9595, 9574-9596, 9574-9598, 9574-9601, 9576-9596,9576-9598, 9576-9601, 9576-9604, 9579-9601, 9579-9604, 9581-9601,9581-9604, and 9583-9604 and (b) the reverse complement of said selectedRNA sequence equivalent to the hepatitis C virus DNA coding strandsequence. In some embodiments, said RNA sequences (a) and (b) are linkedby a loop sequence, and the double-stranded RNA effector molecule(s) isa single RNA strand which forms a stem-loop or hairpin dsRNA structure.In other embodiments, the dsRNA effector molecule(s) is a duplex dsRNAmolecule formed from two separate strands of RNA.

In another aspect, the invention relates to compositions for inhibitingthe expression of a polynucleotide sequence of hepatitis C virus in anin vivo mammalian cell comprising at least one double-stranded RNAeffector molecule, preferably 2, 3, 4, 5, 6 or more double-stranded RNAeffector molecules, or a dsRNA expression construct capable ofexpressing one, 2, 3, 4, 5, 6 or more of said dsRNA effector moleculesin an in vivo mammalian cell, each of said dsRNA effector moleculescomprising (a) a sequence selected from the group consisting of SEQ IDNO: 63; SEQ ID NO: 64; SEQ ID NO: 65; SEQ ID NO:66; SEQ ID NO: 67, SEQID NO: 68, SEQ ID NO: 69; SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQID NO: 73; SEQ ID NO:74; SEQ ID NO:75; and SEQ ID NO:76; (b) the reversecomplement of said selected sequence; and (c) optionally, a sequencelinking sequences (a) and (b); wherein U is substituted for T. Incertain preferred embodiments, the sequence is selected from the groupconsisting of SEQ ID NO:72; SEQ ID NO: 73; SEQ ID NO:74; SEQ ID NO: 75;and SEQ ID NO:76.

In another aspect, the invention relates to methods for inhibiting theexpression of a polynucleotide sequence of hepatitis C virus in an invivo mammalian cell comprising administering at least onedouble-stranded RNA effector molecule, preferably 2, 3, 4, 5, 6 or moredouble-stranded RNA effector molecules, or a dsRNA expression constructcapable of expressing one, 2, 3, 4, 5, 6 or more of said dsRNA effectormolecules in an in vivo mammalian cell, each of said dsRNA effectormolecules comprising (a) a sequence selected from the group consistingof SEQ ID NO: 63; SEQ ID NO: 64; SEQ ID NO: 65; SEQ ID NO:66; SEQ ID NO:67, SEQ ID NO: 68, SEQ ID NO: 69; SEQ ID NO:70, SEQ ID NO:71, SEQ IDNO:72, SEQ ID NO: 73; SEQ ID NO:74; SEQ ID NO:75; and SEQ ID NO:76; (b)the reverse complement of said selected sequence; and (c) optionally, asequence linking sequences (a) and (b); wherein U is substituted for T.In certain preferred embodiments, the sequence is selected from thegroup consisting of SEQ ID NO:72; SEQ ID NO: 73; SEQ ID NO:74; SEQ IDNO: 75; and SEQ ID NO:76.

In another aspect, the invention relates to a polynucleotide sequencecomprising an RNA sequence equivalent to and/or complementary to ahepatitis C virus DNA coding strand sequence selected from the groupconsisting of sequence position 9510-9531, 9510-9533, 9510-9534,9510-9535, 9510-9536, 9514-9534, 9514-9535, 9514-9536, 9514-9539,9514-9540, 9514-9542, 9517-9539, 9517-9540, 9517-9542, 9517-9544,9518-9539, 9518-9540, 9518-9542, 9518-9544, 9520-9540, 9520-9542,9520-9544, 9520-9548, 9521-9542, 9521-9544, 9521-9548, 9521-9549,9522-9542, 9522-9544, 9522-9548, 9522-9549, 9527-9548, 9527-9549,9527-9551, 9527-9552, 9527-9553, 9527-9555, 9528-9548, 9528-9549,9528-9551, 9528-9552, 9528-9553, 9528-9555, 9530-9551, 9530-9552,9530-9553, 9530-9555, 9530-9557, 9530-9558, 9532-9552, 9532-9553,9532-9555, 9532-9557, 9532-9558, 9532-9559, 9532-9560, 9537-9557,9537-9558, 9537-9559, 9537-9560, 9537-9561, 9537-9564, 9538-9558,9538-9559, 9538-9560, 9538-9561, 9538-9564, 9538-9566, 9541-9561,9541-9564, 9541-9566, 9541-9568, 9541-9569, 9543-9564, 9543-9566,9543-9568, 9543-9569, 9543-9571, 9545-9566, 9545-9568, 9545-9569,9545-9571, 9545-9573, 9546-9564, 9546-9566, 9546-9569, 9546-9571,9546-9573, 9547-9568, 9547-9569, 9547-9571, 9547-9573, 9547-9575,9550-9571, 9550-9573, 9550-9575, 9550-9577, 9550-9578, 9554-9575,9554-9577, 9554-9578, 9554-9580, 9556-9577, 9556-9578, 9556-9580,9556-9584, 9562-9584, 9562-9586, 9562-9587, 9562-9588, 9562-9589,9563-9584, 9563-9586, 9563-9587, 9563-9588, 9563-9589, 9563-9591,9565-9586, 9565-9587, 9565-9588, 9565-9589, 9565-9591, 9565-9593,9567-9587, 9567-9588, 9567-9589, 9567-9591, 9567-9593, 9567-9595,9570-9591, 9570-9593, 9570-9595, 9570-9596, 9570-9598, 9572-9593,9572-9595, 9572-9596, 9572-9598, 9574-9595, 9574-9596, 9574-9598,9574-9601, 9576-9596, 9576-9598, 9576-9601, 9576-9604, 9579-9601,9579-9604, 9581-9601, 9581-9604, and 9583-9604.

Applicants' invention further provides a method for inhibitingexpression of a polynucleotide sequence of hepatitis B virus in an invivo mammalian cell comprising administering to said cell adouble-stranded RNA effector molecule comprising an at least 19contiguous base pair nucleotide sequence from within a sequence selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, and SEQ ID NO:10; wherein U is substituted for T. In a preferredembodiment of the method, effector sequences from more than one SEQ IDsequence may be administered to the same cell, and/or more than oneeffector sequence from within the same SEQ ID sequence may beadministered to the same cell.

Applicants further provide a method for inhibiting expression of apolynucleotide sequence of hepatitis C virus in an in vivo mammaliancell comprising administering to said cell a double-stranded RNAeffector molecule comprising an at least 19 contiguous base pairnucleotide sequence from within a sequence selected from the groupconsisting of SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:27; wherein U issubstituted for T. In a preferred embodiment of this aspect of themethod, effector molecules from both SEQ ID NO:11 and SEQ ID NO:12 maybe administered to the same cell; or from both SEQ ID NO: 11 and SEQ IDNO:27; or from both SEQ ID NO: 12 and SEQ ID NO:27; or from each of SEQID NO: 11, SEQ ID NO:12, and SEQ ID NO:27, are administered to the samecell; and/or more than one effector molecule from within the same SEQ IDNO may be administered to the same cell.

Applicants further provide a method for inhibiting expression of both apolynucleotide sequence of hepatitis B virus and a polynucleotidesequence of hepatitis C virus in the same in vivo mammalian cell,comprising administering to said cell a double-stranded RNA effectormolecule comprising a first at least 19 contiguous base pair nucleotidesequence from within a sequence selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10; wherein Uis substituted for T; and a double-stranded RNA effector moleculecomprising a second at least 19 contiguous base pair nucleotide sequencefrom within a sequence selected from the group consisting of SEQ IDNO:11, SEQ ID NO:12, and SEQ ID NO:27; wherein U is substituted for T.In preferred embodiments of this aspect of the invention, effectormolecules from more than one of SEQ ID NO:1 through SEQ ID NO:10 may beadministered to the same cell; and/or effector molecules from both SEQID NO:11 and SEQ ID NO:12; or from both SEQ ID NO: 11 and SEQ ID NO:27;or from both SEQ ID NO: 12 and SEQ ID NO:27; or from SEQ ID NO: 11, SEQID NO:12 and SEQ ID NO:27; may be administered to the same cell; and/ormore than one effector molecules from within the same SEQ ID NO may beadministered to the same cell.

Applicants further provide a composition for inhibiting the expressionof a polynucleotide sequence of hepatitis B virus in an in vivomammalian cell comprising a double-stranded RNA effector moleculecomprising an at least 19 contiguous base pair nucleotide sequence fromwithin a sequence selected from the group consisting of SEQ ID NO:1, SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10; wherein U issubstituted for T. Preferred embodiments of the composition includewherein effector molecules from more than one of SEQ ID NO:1 through SEQID NO:10 are present in the composition; and/or wherein more than oneeffector molecule from within the same SEQ ID NO is present in thecomposition.

Applicants further provide a composition for inhibiting the expressionof a polynucleotide sequence of hepatitis C virus in an in vivomammalian cell comprising a double-stranded RNA effector moleculecomprising an at least 19 contiguous base pair nucleotide sequence fromwithin a sequence selected from the group consisting of SEQ ID NO:11 andSEQ ID NO:12 and SEQ ID NO:27; wherein U is substituted for T. Preferredembodiments of the composition include wherein effector molecules fromboth SEQ ID NO:11 and SEQ ID NO:12 are present in the composition; orfrom both SEQ ID NO: 11 and SEQ ID NO:27; or from both SEQ ID NO: 12 andSEQ ID NO:27; or from each of SEQ ID NO: 11, SEQ ID NO:12, and SEQ IDNO:27, are present in the same composition, and/or wherein more than oneeffector molecule from within the same SEQ ID NO may be present in thecomposition.

Applicants further provide a composition for inhibiting the expressionof both a polynucleotide sequence of hepatitis B virus and apolynucleotide sequence of hepatitis C virus in a single in vivomammalian cell comprising a double-stranded RNA effector moleculecomprising a first at least 19 contiguous base pair nucleotide sequencefrom within a sequence selected from the group consisting of SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10; wherein U issubstituted for T; and a double-stranded RNA effector moleculecomprising a second at least 19 contiguous base pair nucleotide sequencefrom within a sequence selected from the group consisting of SEQ IDNO:11 and SEQ ID NO:12 and SEQ ID NO:27; wherein U is substituted for T.Preferred embodiments of the composition include wherein effectormolecules from more than one of SEQ ID NO:1 through SEQ ID NO:10 arepresent in the composition; and/or wherein effector molecules from bothSEQ ID NO:11 and SEQ ID NO:12; or from both SEQ ID NO: 11 and SEQ IDNO:27; or from both SEQ ID NO: 12 and SEQ ID NO:27; or from each of SEQID NO: 11, SEQ ID NO:12, and SEQ ID NO:27, are present in thecomposition; and/or wherein more than one effector sequence from withinthe same SEQ ID NO may be present in the composition.

In particularly preferred embodiments of the above methods andcompositions of the invention, the polynucleotide sequence is presentwithin a double-stranded region of an RNA, and the mammalian cell is ahuman cell.

Further provided are compositions for inhibiting the expression of apolynucleotide sequence of hepatitis B virus and/or a polynucleotidesequence of hepatitis C virus in mammalian cells, wherein saidcompositions comprise an at least 19 contiguous nucleotide sequenceselected from within SEQ ID NO:1 through SEQ ID NO:12, and SEQ ID NO:27;the complement sequences of said SEQ ID NO:1 through SEQ ID NO:12, andSEQ ID NO: 27 sequences, and mixtures of these sequences. In thisembodiment of the invention, the “an at least 19 contiguous nucleotidesequence” is preferably DNA, and the mammalian cell is preferably human.Also provided are expression constructs comprising any of theaforementioned compositions and a mammalian cell comprising saidexpression constructs.

Another aspect provides for a polynucleotide sequence comprising asequence selected from SEQ ID NO:14 through SEQ ID NO:26. Another aspectof the invention provides for polynucleotide sequence comprisingnucleotides 1-19, 1-20, 1-21, 2-20, 2-21, or 3-21 of a sequence selectedfrom SEQ ID NO:14 through SEQ ID NO:26. Another aspect of the inventionprovides for a polynucleotide sequence comprising an at least 19contiguous base pair nucleotide sequence from within a sequence selectedfrom SEQ ID NO:27 through SEQ ID NO:44.

Another aspect provides a composition for inhibiting the expression of apolynucleotide sequence of hepatitis C virus in a mammalian cell,comprising a double-stranded RNA effector molecule comprising an atleast 19 contiguous base pair nucleotide sequence from within SEQ IDNO:27; wherein U is substituted for T.

In various aspects of the foregoing methods and compositions, the invivo mammalian cell is an in vivo human cell.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 through SEQ ID NO:10 represent conserved regions of thehepatitis B genome.

SEQ ID NO:11 and SEQ ID NO:12 represent conserved regions of thehepatitis C genome.

SEQ ID NO:13 represents the nucleotide sequence of human U6 promoter.

SEQ ID NO:14 and SEQ ID NO:15 represent eiRNAs that have HBV sequencesmapping within SEQ ID NO:5.

SEQ ID NO:16 and SEQ ID NO:17 represent eiRNAs that have HBV 20sequences mapping within SEQ ID NO:4.

SEQ ID NO:18 represents eiRNA that has an HBV sequence mapping withinSEQ ID NO:10.

SEQ ID NO:19 through SEQ ID NO:22 represent eiRNAs that have HBVsequences mapping within SEQ ID NO:3.

SEQ ID NO:23 and SEQ. ID NO:24 represent eiRNAs that have HBV sequencesmapping within SEQ ID NO:2.

SEQ ID NO:25 and SEQ ID NO:26 represent eiRNAs that have HBV sequencesmapping within SEQ ID NO:1.

SEQ ID NO:27 represents the “X” region of the HCV 3′UTR.

SEQ ID NO:28 through SEQ ID NO:36 represent siRNAs mapping to the HCV3′UTR.

SEQ ID NO:37 through SEQ ID NO:44 represent siRNAs mapping to the “X”region of the HCV 3′UTR.

SEQ ID NO:45 represents an siRNA mapping to the HCV core.

SEQ ID NO:46 represents an siRNA mapping to lamin.

SEQ ID NO:47 represents the T7 RNA polymerase gene.

SEQ ID NO:48 represents a 5′ segment of the hepatitis C virus sequence(corresponds to positions 36 to 358 in Genbank Accession NumberAJ238799, with 2 base changes, C to G at AJ238799 position 204 and G toA at AJ238799 position 357).

SEQ ID NO:49 represents an eiRNA (shRNA) molecule to a conserved HBVsequence.

SEQ ID NO:50 through SEQ ID NO:62 represent the first 21 nucleotides ofSEQ ID NOs: 14-23, 25-26, and 49.

SEQ ID NO:63 through SEQ ID NO:71 represent the first 21 nucleotides ofSEQ ID NOs: 28-36.

SEQ ID NO:72 through SEQ ID NO:76 represent highly conserved codingregion sequitopes from the 5′ and 3′ untranslated regions of HCV.

SEQ ID NO:77 through SEQ ID NO:109 represent highly conserved HCVsequences from the 5′ UTR of the HCV (SEQ ID NO: 11).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a vector illustrating placement of the T7 RNA polymerasepromoter and T7 RNA polymerase, showing inclusion of hairpin eiRNAsequences.

FIG. 2 is a graph showing HBsAg inhibition corresponding to data 15found in Table 2.

FIG. 3 is a graph showing HBsAg inhibition corresponding to data foundin Table 3.

FIG. 4 is a graph showing HBsAg inhibition corresponding to data foundin Table 4.

FIG. 5 is a graph showing HBsAg inhibition corresponding to data foundin Table 5.

FIG. 6 is a graph showing HBsAg inhibition corresponding to data foundin Table 6.

FIG. 7 is a graph showing HBsAg inhibition corresponding to data foundin Table 7.

FIG. 8 is a graph showing HBsAg inhibition corresponding to data foundin Table 8.

FIG. 9 is a drawing depicting effective HBV-AYW shRNA inserts.

FIG. 10 is a graph showing HBsAg inhibition corresponding to data foundin Table 9.

FIG. 11 is a bar graph showing downregulation of HBV RNA by NorthernBlot analysis.

FIG. 12 is a graph showing HBsAg inhibition corresponding to data foundin Table 12.

FIG. 13 is a Western Blot showing levels of HCV NS5A protein at (1 to r)0, 9, and 20 pmole of the identified siRNAs, as described in more detailin Experiment 1 of Example 2.

FIG. 14 is a Western Blot showing levels of HCV NS5A protein at 20 (I tor) 0, 9, and 20 pmole of the identified siRNA, and 0, 3, and 9 pmole ofthe “core” positive control siRNA, as described in more detail inExperiment 2 of Example 2.

FIG. 15 is a table of additional conserved HCV genome sequence segmentssuitable for generating dsRNA effector molecules which inhibit theexpression of polynucleotide sequences of hepatitis C virus, includingexpressed shRNA for gene silencing. Each sequence represents a DNAcoding strand sequence in standard 5′ to 3′ polarity which (togetherwith its reverse complement) can be utilized to transcribe or design adouble-stranded RNA effector molecule, e.g., an shRNA or duplex dsRNAmolecule targeted to degrade the negative strand of HCV RNA. E.g., anDNA sequence, followed by a loop sequence (e.g., a 9 base loop sequenceas described elsewhere herein), followed by the reverse complement ofthe sequence given in the table, may be incorporated into an expressionconstruct under the control of an appropriate promoter. The shRNAmolecule transcribed from such an expression construct is expected toinhibit expression of HCV polynucleotide sequences and/or mediate dsRNAsilencing of HCV. For example, in the case of the 22 base sequence shownfor positions 9545-9566, a construct is made to contain a 53 by insert,comprising the 22 base sequence of 9545-9566, a linker or loop sequence,and the reverse complement of the 9545-9566 sequence, preferably underthe control of an RNA polymerase III promoter and ending with an RNApolymerase III terminator, e.g., a run of 4, 5, or more T residues. TheRNA equivalent of this sequence, having U's instead of T's, would read(in the 5′ to 3′ direction):

AAAGGUCCGUGAGCCGCUUGAC-XXXXXXXXX- GUCAAGCGGUCACGGACCUU U

where X represents bases of the loop that are unable to form stable basepairs with any other portion of the 53 by shRNA sequence. The loop mayvary considerably, however, as to both length and nucleotide sequence,so long as the formation of the double-stranded “stem” region of thehairpin is not adversely affected. Thus, in expression constructs thatare the subject of this invention, the sequence element above beginningat the end which reads 5′ AAAGGT is cloned into an appropriate vectordownstream from and operably linked to the promoter. As describedelsewhere herein, in preferred embodiments, two, three, four, five, six,seven, or more of the shRNAs encoded by these sequences, optionally,together with other anti-HCV, and/or HBV sequences described herein, arecoded into and expressed by a single dsRNA expression vector. In oneaspect, each of said multiple stem-loop or shRNA molecules is encoded ina single expression vector within a different expression cassette, eachoperably linked to a promoter and a terminator, preferably a polymeraseIII promoter, which may be the same or different. In another aspect, twoor more hairpin dsRNA molecules may be expressed from a single promoter,as e.g., a bi-fingered molecule in which a single transcribed RNA strandcomprises two such shRNA sequences separated by an unrelated linkersequence. Such constructs, in which a single expression vector providesa mammalian cell with two, three, four, five or more independent dsRNAeffector molecules against an HCV and/or HBV target polynucleotide, areparticularly desirable for pharmaceutical applications. An alternativemeans of dsRNA-mediated silencing may be accomplished by preparingshRNAs or duplex dsRNAs corresponding to the identified sequences bychemical synthesis or in vitro expression and delivering them into acell in order to achieve inhibition of HCV and/or HBV polynucleotidesequences.

DETAILED DESCRIPTION OF THE INVENTION

RNA interference (RNAi) is the process of sequence-specific,post-transcriptional gene silencing or transcriptional gene silencing inanimals and plants, initiated by double-stranded RNA (dsRNA) that ishomologous in sequence to the silenced gene. Since RNA interference actsin a sequence specific manner, the RNAi molecule used as a drug must bespecific to its target. Viral genomes are variable to accommodateresistance to changes in the environment. While HBV and HCV are verydesirable viral targets for RNAi, the variability and mutability of theviruses and the high rates of transcription of the viruses make HBV andHCV very challenging targets for any therapeutic and/or prophylacticapproach. In order to knock down viral genome replication using RNAithere is a need to identify conserved and unique regions in the viralgenome. At that same time, it is very important in order to avoidtoxicity that any sequences selected for gene silencing be absent fromthe human genome.

Human Hepatitis B Virus (HBV)

Hepatitis B virus belongs to the family of hepadnaviruses. The HBVgenome is a relaxed circular, partially double stranded DNA ofapproximately 3,200 base pairs. There are 4 partially overlapping openreading frames encoding the envelope (pre-S/S), core (precore/core),polymerase, and X proteins. The pre-S/S open reading frames encode thelarge (L), middle (M), and small (S) surface glycoproteins. Theprecore/core open reading frame is translated into a precorepolypeptide, which is modified into a soluble protein, the hepatitis B eantigen (HBeAg) and the nucleocapsid protein, hepatitis B core antigen.Mutations in the core promoter and precore region have been shown todecrease or abolish HBeAg production. The polymerase protein functionsas a reverse transcriptase as well as a DNA polymerase. The X protein isa potent transactivator and may play a role in hepatocarcinogenesis.

The replication cycle of HBV begins with the attachment of the virion tothe hepatocyte. Inside the hepatocyte nucleus, synthesis of the plusstrand HBV DNA is completed and the viral genome is converted into acovalently closed circular DNA (cccDNA). Most antiviral agents that havebeen examined so far have little or no effect on cccDNA, which accountsfor the rapid reappearance of serum HBV DNA after cessation of antiviraltherapy. The aims of treatment of chronic hepatitis B are to achievesustained suppression of HBV replication and/or expression of HBVantigens and remission of liver disease.

In GenBank version 132.0 there are more then 4500 HBV sequences and 340HBV complete genome sequences (317 Human isolates, 22 isolates fromother primates and one woodchuck HBV isolate). This variabilityconstitutes a serious challenge for sequence-specific pharmaceuticalapproaches such as RNAi. In order to identify conserved sequencessuitable for RNAi applications, a comparison between all the completegenomes was carried out using a modified version of ClustalW. Twomultiple alignment schemes were generated: the first included all 339HBV complete genome sequences and the second was limited to all HumanHBV isolates. The multiple alignment results were parsed and a tablethat included scores for sequence conservation at each position in theHBV genome was generated. A sliding window search to identify thelongest region of sequence conservation larger then 19 nt in length wascreated. Three major conserved regions were identified and mapped toGenBank accession no.: AF090840, a Human HBV isolate. The conserved HBVsequences were screened against Genbank sequences of both human genomicand cDNA libraries (Human chromosomes database). It was found that 21nucleotide and longer segments selected as a permuted “window” fromwithin the conserved regions were unique to HBV, i.e. no perfectsequence matches exist between any 21 nt or longer HBV conservedsegments and the available sequence databases of human chromosomal andRNA sequences. For human therapeutic purposes, assuring that homologoushuman sequences are not inadvertently silenced is as important asselecting conserved viral sequences for RNAi.

Human Hepatitis C Virus

HCV is a small (40 to 60 nanometers in diameter), enveloped,single-stranded RNA virus of the family Flaviviridae and genushepacivirus. The genome is approximately 10,000 nucleotides and encodesa single polyprotein of about 3,000 amino acids, which ispost-transcriptionally cleaved into 10 polypeptides, including 3 majorstructural (C, E1, and E2) and multiple non-structural proteins ([NS]NS2 to NS5). The NS proteins include enzymes necessary for proteinprocessing (proteases) and viral replication (RNA polymerase). Becausethe virus mutates rapidly, changes in the envelope proteins may help itevade the immune system. There are at least 6 major genotypes and morethan 90 subtypes of HCV. The different genotypes have differentgeographic distributions. Genotypes 1a and 1b are the most common in theUnited States (about 75% of cases). Genotypes 2a and 2b (approximately15%) and 3 (approximately 7%) are less common.

There is little difference in the severity of disease or outcome ofpatients infected with different genotypes. However, patients withgenotypes 2 and 3 are more likely to respond to interferon treatment.The virus replicates at a high rate in the liver and has marked sequenceheterogeneity. The main goal of treatment of chronic hepatitis C is toeliminate detectable viral RNA from the blood. Lack of detectablehepatitis C virus RNA from blood six months after completing therapy isknown as a sustained response. Studies suggest that a sustained responseis equated with a very favorable prognosis and that it may be equivalentto a cure. There may be other more subtle benefits of treatment, such asslowing the progression of liver scarring (fibrosis) in patients who donot achieve a sustained response.

In GenBank version 134.0 there are more then 20,000 HCV sequences and 93HCV complete genome sequences. A comparison between all the completegenomes was carried out using a modified version of ClustalW. Themultiple alignment result was parsed and a table that included scoresfor sequence conservation at each position in the HCV genome wasgenerated. A sliding window search to identify the longest region ofsequence conservation larger then 19 nt in length was created. Threemajor conserved regions were identified and mapped to GenBank RefSeq(reference sequence) accession no.: NC_004102 this is GenBank annotatedHCV complete genome. The three major conserved regions include a portionof the 3′ untranslated region of the virus, already described in theliterature to be well-conserved among viral isolates. See, e.g., U.S.Pat. No. 5,874,565, “Nucleic Acids Comprising a Highly Conserved Novel3′ Terminal Sequence Element of the Hepatitis C Virus.” However, theinstant disclosure represents a comprehensive and detailed analysis ofthese conserved regions to the extent that permitted the discovery andevaluation of multiple short segments suitable for use alone and incombination as a therapeutic for silencing HCV among a diverse patientpopulation. The conserved sequences were screened against Genbanksequences of both human genomic and cDNA libraries (human chromosomesdatabase), and the series of permuted HCV segments greater than 20 baseslong with no homology to the human sequence databases were identified.

Non-Homology with Human Sequences

It is equally important to ensure that conserved viral sequencestargeted for silencing according to the invention be substantiallynon-homologous to any naturally occurring, normally functioning, andessential human polynucleotide sequence, so that the dsRNA molecule doesnot adversely affect the function of any essential naturally occurringmammalian polynucleotide sequence, when used in the methods of thisinvention. Such naturally occurring functional mammalian polynucleotidesequences include mammalian sequences that encode desired proteins, aswell as mammalian sequences that are non-coding, but that provide foressential regulatory sequences in a healthy mammal. Essentially, the RNAmolecule useful in this invention must be sufficiently distinct insequence from any mammalian polynucleotide sequence for which thefunction is intended to be undisturbed after any of the methods of thisinvention are performed. Computer algorithms may be used to define theessential lack of homology between the RNA molecule polynucleotidesequence and the normal mammalian sequences.

Since the length of a contiguous dsRNA sequence capable of associationwith and activation of RISC (RNA-induced silencing complex), isgenerally considered to be 19-27 base pairs, the identified conservedHBV and HCV sequences were compared with both human genomic librariesand, perhaps even more importantly, with human cDNA libraries asdescribed above. Since human cDNA libraries represent expressedsequences that appear in mRNAs, such mRNA sequences would be especiallyvulnerable to silencing by homologous dsRNA sequences provided to acell.

Accordingly, the conserved HBV and HCV sequences were compared withhuman genomic and cDNA sequences. No human cDNA library matches to theHBV or HCV conserved sequences were identified. (Although there weresome matches that were ultimately identified as HBV contamination in thecDNA library.) A comparison with human genomic library sequencesrevealed no match of any sequence of 21 nts or more, one match of 20nucleotides, and one match of 19 nucleotides. These matches were innon-coding regions, and likely do not appear in mRNA since cognates werenot turned up in the cDNA library. Therefore, they are consideredunlikely to be a safety risk, but could be excluded if desired.

Conserved Sequences from HBV and HCV

HBV Conserved Region 1 GAACATGGAGA[A(89%)/G(11%)]CA[T(76%)/C(24%)][C(78%)/A(20%)/T(2%)][A(78%)/G(21%)/T(1%)1CATCAGGA[T(65%)/c(35%)]TCCTAGGACCCCTGCTCGTGTTACAGGCGG[G(88%)/t(12%)]GT[T(89%)/G(11%)]TTTCT[T(94%)/C(6%)]GTTGACAA[G(64%)/A(36%)]AATCCTCACAATACC[A(56%)/G(43%)/T(1%)]CAGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGG[G(92%)/A(5%)/T(3%)]A[A(41%)/G(30%)/T(18%)/C(11%)][C(90%)/T(10%)] HBV Conserved Region 2TGGATGTGTCT[G(99%)/A(1%)]CGGCGTTTTATCAT HBV Conserved Region 3AAGGCCTTTCT[A(43%)/G(43%)/C(14%)][T(56%)/A(37%)/C(7%)]GT[A(87%)/C(13%)]AACA[A(57%)/G(43%)]TA[T(59%)/C(41%)][C(59%)/A(41%)]TG[A(92%)/C(8%)][A(93%)/C(7%)]CaTTTACCCCGTTGC[T(54%)/C(46%)][C(92%)/A(8%)]GGCAACGG[C(74%)/T(24%)]C[A(50/T(43%)/c(7%)]GG[T(87%)/C(13%)]CT[G(70%)/C(19%)/T(7%)/A(4%)]TGCCAAGTGTTTGCTGACGCAACCCCCACTGG[C(48%)/T(38%)/A(14%)]TGGGGCTTGG[C(84%)/T(16%)][C(84%)/T(12%)/G(4%)]AT[A(47%)/T(23%)/G(17%)/C(13%)]GGCCATC[A(83%)/G(17%)][G(92%)/C(8%)]CGCATGCGTGGAACCTTT[G(84%)/C(13%)/T(3%)][T(92%)/A(4%)/C(3%)/G(1%)]G[G(78%)/T(22%)]CTCCTCTGCCGATCCATACTGCGGAACTCCT[A(88%)/T(9%)/G(1%)/C(1%)]GC[C(57%)/A(35%)/T(6%)/G(2%)]GC[T(92%)/C(7%)/G(1%)]TGTTT[T(88%)/C(12%)]GCTCGCAGC[C(64%)/A(36%)1GGTCTGG[A(87%)/G(13%)]GC HBV Conserved Region 4[C(62%)/T(38%)]ACTGTTCAAGCCTCAAGCTGTGCCTTGGGTGGCTTT[G(88%)/A(12%)]GG[G(92%)/A(8%)]CATGGACATTGAC[C(92%)/A(8%)]C[T(65%)/G(35%)]TATAAAGAATTTGGAGCT[A(65%)/T(35%)]CTGTGGAGTTACTCTC[G(62%)/T(35%)/A(3%)]TTTTTGCCTTC[T(92%)/C(8%)]GACTT[C(92%)/ T(8%)]TTTCCTTCHBV Conserved Region 5 [C(69%)/del(31%)1[G(69%)/del(31%)]A[G(85%)/T(11%)/C(4%)]GCAGGTCCCCTAGAAGAAGAACTCCCTCGCCTCGCAGACG[C(61%)/A(39%)1AG[A(62%)/G(38%)]TCTCAATCG[C(88%)/A(12%)]CGCGTCGCAGAAGATCTCAAT[C(92%)/T(8%)]TCGGGAATCT[C(88%)/T(12%)]AATGTTAGTAT HBV Conserved Region 6TTGG[C(84%)/t(16%)][C(84%)/t(12%)/g(4%)]AT[A(47%)/t(23%)/g(17%)/c(13%)]GGCCATC[A(83%)/g(17%)][G(92%)/c(8%)]CGCATGCGTGGAACCTTT[G(84%)/c(13%)/t(3%)][T(92%)/a(4%)/c(3%)/g(1%)]G[G(78%)/t(22%)]CTCCTCTGCCGATCCATACTGCGGAACTCCT[A(88%)/t(9%)/g(1%)/c(1%)]GC[C(57%)/a(35%)/t(6%)/g(2%)]GC[T(92%)/c(7%)/g(1%)]TGTTT[T(88%)/c(12%)]GCTCGCAGC[C(64%)/a(36%)]GGTCTGG[A(87%)/g(13%)]GC HBV Conserved Region 7CTGCCAACTGGAT[C(86%)/T(10%)/A(4%)]CT[C(69%)/T(25%)/A(6%)]CGCGGGACGTCCTTTGT[T(75%)/C(25%)TACGTCCCGTC[G(93%)/A(7%)]GCGCTGAATCC[C(86%)/T(7%)/A(7%)]GCGGACGACCC[C(52%)/G(25%)/T(19%)/ A(4%)]HCV Conserved Region 1 [A(74%)/G(19%)/T(7%)][G(82%)/A(15%)/T(3%)]ATCACTCCCCTGTGAGGAACTACTGTCTTCACGCAGAAAGCGTCTAGCCATGGCGTTAGTATGAGTGT[C(92%)/T(7%)]GTGCAGC[C(89%)/T(10%)]TCCAGG[A(76%)/T(14%)/C(8%)/G(1%)]CCCCCCCTCCCGGGAGAGCCATAGTGGTCTGCGGAACCGGTGAGTACACCGGAATTGCC[A(90%)/G(9%)]GGA[C(78%)/T(16%)/A(5%)]GACCGGGTCCTTTCTTGGAT[G(78%)/T(11%)/A(10%)]AACCCGCTC[A(94%)/T(5%)]ATGCC[T(90%)/C(9%)]GGA[G(91%)/C(4%)/A(4%)]ATTTGGGCGTGCCCCCGC[G(85%)/A(14%)]AGAC[T(94%)/C(5%)]GCTAGCCGAGTAG[T(92%)/C(7%)]GTTGGGT[C(94%)/T(5%)]GCGAAAGGCCTTGTGGTACTGCCTGATAGGGTGCTTGCGAGTGCCCCGGGAGGTCTCGTAGACCGTGCA[C(62%)/T(30%)/A(8%)]CATGAGCAC[A(50%)/G(50%)][A(92%)/C(8%)][A(89%)/T(11%)]TCC[T(92%)/A(5%)/C(3%)]AAACC[T(84%)/C(14%)/A(2%)]CAAAGAAAAACCAAA[C(84%)/A(16%)]G[T(84%)/A(16%)]AACACCAACCG[C(77%)/T(23%)]CGCCCACAGGACGT[C(81%)/T(18%)/A(1%)]AAGTMCCGGG[C(89%)/T(11%)GG[T(80%)/C(20%)]GG[T(80%)/C(17%)/A(3%)]CAGATCGTTGG[T(91%)/C(8%)/G(1%)]GGAGT[T(87%)/A(11%)/C(2%)]TAC[C(74%)/T(20%)/G(6%)]TGTTGCCGCGCAGGGGCCC[C(87%)/T(8%)/A(4%)/G(1%)][A(92%)/C(8%)][G(92%)/A(5%)/C(2%)][G(87%)/A(12%)/T(1%)]TTGGGTGTGCGCGCGAC[T(78%)/G(13%)/A(7%)/C(2%)]AGGAAGACTTC[C(90%)/G(5%)/T(5%)]GA[G(90%)/A(10%)]CGGTC[G(79%)/C(12%)/A(8%)/T(1%)]CA[A(86%)/G(14%)]CC[T(88%)/A(6%)C(6%)]CG[T(82%)/C(9%)A(9%)]GG[A(87%)/T(8%)/G(3%)/C(2%)] AGHCV Conserved Region 2ATGGC[T(76%)/A(12%)/C(10%)/G(2%)]TGGGATATGATGATGAA CTGG[T(81%)/C(19%)]C

Conserved Consensus Sequences Presented in SEQ ID Format

The following sequences are presented in the format required per theWIPO Standard ST.25 (1998), using the codes provided under 37 CFR 1.821.SEQ ID NO:1 through SEQ ID NO:10 are derived from the HBV genome SEQ IDNO:11 and SEQ ID NO:12 are derived from the HCV genome.

HBV SEQ ID NO: 1 GAACATGGAGArCAyhdCATCAGGAyTCCTAGGACCCCTGCTCGTGTTACAGGCGGkGTkTTTCTyCTTGACAArAATCCTCACAATACCdCAGAGTCTAGACTCGTGGTGGACTTCTCTCAATTTTCTAGGGGdAny HBV SEQ ID NO: 2TGGATGTGTCTrCGGCGTTITATCAT HBV SEQ ID NO: 3AAGGCCTTTCTvhGTmAACArTAymTGmmCCTTTACCCC GTTGCymGGCAACGGyehGGyCTnTGCCAAGTGTTTGCTGACGCAACCCCCACTGGhTGGGGCTTGGybATnGGCCATCrsCGCATGCGTGGAACCTTTbnGkCTCCTCTGCCGATCCATACTGCGGAACTCCTnGCnGCbTGTTTyGCTCGCAGCmGGTCTGGrGC HBV SEQ ID NO: 4yACTGTTCAAGCCTCAAGCTGTGCCTTGGGTGGCTTTrGGrCATGGACATTGACmCkTATAAAGAATTTGGAGCTwCTGTGGAGTTACTCTCdTTTTTGCCTTCyGACTTyTTTCCTTC HBV SEQ ID NO: 5CGAbGCAGGTCCCCTAGAAGAAGAACTCCCTCGCCTCGCAGACGmAGrTCTCAATCGmCGCGTCGCAGAAGATCTCAATyTCGGGAATCTyAAT GTTAGTAT HBVSEQ ID NO: 6 AbGCAGGTCCCCTAGAAGAAGAACTCCCTCGCCTCGCAGACGmAGrTCTCAATCGmCGCGTCGCAGAAGATCTCAATyTCGGGAATCTyAATGT TAGTAT HBVSEQ ID NO: 7 CAbGCAGGTCCCCTAGAAGAAGAACTCCCTCGCCTCGCAGACGmAGrTCTCAATCGmCGCGTCGCAGAAGATCTCAATyTCGGGAATCTyAATG TTAGTAT HBVSEQ ID NO: 8 GAbGCAGGTCCCCTAGAAGAAGAACTCCCTCGCCTCGCAGACGmAGrTCTCAATCGmCGCGTCGCAGAAGATCTCAATyTCGGGAATCTyAATG TTAGTAT HBVSEQ ID NO: 9 TTGGybATnGGCCATCrsCGCATGCGTGGAACCTTTbnGkCTCCTCTGCCGATCCATACTGCGGAACTCCTnG CnGCbTGTTTyGCTCGCAGCmGGTCTGGrGC HBVSEQ ID NO: 10 CTGCCAACTGGAThCThCGCGGGACGTCCTTTGTyTACGTCCCGTCrGCGCTGAATCChGCGGACGACCCn HCV SEQ ID NO: 11DdATCACTCCCCTGTGAGGAACTACTGTCTTCACGCAGAAAGCGTCTAGCCATGGCGTTAGTATGAGTGTyGTGCAGCyTCCAGGnCCCCCCCTCCCGGGAGAGCCATAGTGGTCTGCGGAACCGGTGAGTACACCGGAATTGCCrGGAhGACCGGGTCCTTTCTTGGATdAACCCGCTCwATGCCyGGAvATTTGGGCGTGCCCCCGCrAGACyGCTAGCCGAGTAGyGTTGGGTyGCGAAAGGCCTTGTGGTACTGCCTGATAGGGTGCTTGCGAGTGC CCCGGGAGGTCTCGTAGACCGTGCAhCATGAGCACrmwTCChAAACChCAAAGAAAAACCAAAmGwAACACCAACCGyCGCCCACAGGACGThAAGTTCCCGGGyGGyGGhCAGATCGTTGGbGGAGThTACbTGTTGCCGCGCAGGGGCCCnmvdTTGGGTGTGCGCGCGACnAGGAAGACTTCbGArCGGTCnCArCChCGhGGnAG

Double Stranded RNA Gene Silencing/RNAi

By “nucleic acid composition” or “nucleotide” composition is meant anyone or more compounds in which one or more molecules of phosphoric acidare combined with a carbohydrate (e.g., pentose or hexose) which are inturn combined with bases derived from purine (e.g., adenine) and frompyrimidine (e.g., thymine). Particular naturally occurring nucleic acidmolecules include genomic deoxyribonucleic acid (DNA) and hostribonucleic acid (RNA), as well as the several different forms of thelatter, e.g., messenger RNA (mRNA), transfer RNA (tRNA), and ribosomalRNA (rRNA). Also included are different DNA molecules which arecomplementary (cDNA) to the different RNA molecules. Synthesized DNA ora hybrid thereof with naturally occurring DNA, as well as DNA/RNAhybrids, and peptide nucleic acid (PNA) molecules (Gambari, Curr PharmDes 2001 November; 7(17):1839-62) can also be used.

It is contemplated that where the desired nucleic acid molecule is RNA,the T (thymine) in the sequences provided herein is substituted with U(uracil). For example, SEQ ID NO:1 through SEQ ID NO:44 are disclosedherein as DNA sequences. It will be obvious to one of ordinary skill inthe art that an RNA effector molecule comprising sequences from any ofthe aforementioned SEQ ID NOs will have T substituted with U.

Nucleic acids typically have a sequence of two or more covalently bondednaturally-occurring or modified deoxyribonucleotides or ribonucleotides.Modified nucleic acids include, e.g., peptide nucleic acids andnucleotides with unnatural bases.

By “dsRNA” or “dsRNA effector molecule” is meant a nucleic acidcontaining a region of two or more nucleotides that are in a doublestranded conformation. It is envisioned that the conserved viralsequences of the invention may be utilized in any of the manycompositions of “dsRNA effector molecules” known in the art orsubsequently developed which act through a dsRNA-mediated gene silencingor RNAi mechanism, including, e.g., “hairpin” or stem-loopdouble-stranded RNA effector molecules in which a single RNA strand withself-complementary sequences is capable of assuming a double-strandedconformation, or duplex dsRNA effector molecules comprising two separatestrands of RNA. In various embodiments, the dsRNA consists entirely ofribonucleotides or consists of a mixture of ribonucleotides anddeoxynucleotides, such as the RNA/DNA hybrids disclosed, for example, byWO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filedApr. 21, 1999. The dsRNA or dsRNA effector molecule may be a singlemolecule with a region of self-complementarity such that nucleotides inone segment of the molecule base pair with nucleotides in anothersegment of the molecule. In various embodiments, a dsRNA that consistsof a single molecule consists entirely of ribonucleotides or includes aregion of ribonucleotides that is complementary to a region ofdeoxyribonucleotides. Alternatively, the dsRNA may include two differentstrands that have a region of complementarity to each other. In variousembodiments, both strands consist entirely of ribonucleotides, onestrand consists entirely of ribonucleotides and one strand consistsentirely of deoxyribonucleotides, or one or both strands contain amixture of ribonucleotides and deoxyribonucleotides. Desirably, theregions of complementarity are at least 70, 80, 90, 95, 98, or 100%complementary to each other and to a target nucleic acid sequence.Desirably, the region of the dsRNA that is present in a double strandedconformation includes at least 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 50, 75, 100, 200, 500, 1000, 2000 or 5000 nucleotides orincludes all of the nucleotides in a cDNA or other target nucleic acidsequence being represented in the dsRNA. In some embodiments, the dsRNAdoes not contain any single stranded regions, such as single strandedends, or the dsRNA is a hairpin. In other embodiments, the dsRNA has oneor more single stranded regions or overhangs. Desirable RNA/DNA hybridsinclude a DNA strand or region that is an antisense strand or region(e.g, has at least 70, 80, 90, 95, 98, or 100% complementarity to atarget nucleic acid) and an RNA strand or region that is a sense strandor region (e.g, has at least 70, 80, 90, 95, 98, or 100% identity to atarget nucleic acid), and vice versa. In various embodiments, theRNA/DNA hybrid is made in vitro using enzymatic or chemical syntheticmethods such as those described herein or those described in WO00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr.21, 1999. In other embodiments, a DNA strand synthesized in vitro iscomplexed with an RNA strand made in vivo or in vitro before, after, orconcurrent with the transformation of the DNA strand into the cell. Inyet other embodiments, the dsRNA is a single circular nucleic acidcontaining a sense and an antisense region, or the dsRNA includes acircular nucleic acid and either a second circular nucleic acid or alinear nucleic acid (see, for example, WO 00/63364, filed Apr. 19, 2000,or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999.) Exemplary circularnucleic acids include lariat structures in which the free 5′ phosphorylgroup of a nucleotide becomes linked to the 2′ hydroxyl group of anothernucleotide in a loop back fashion.

In other embodiments, the dsRNA includes one or more modifiednucleotides in which the 2′ position in the sugar contains a halogen(such as fluorine group) or contains an alkoxy group (such as a methoxygroup) which increases the half-life of the dsRNA in vitro or in vivocompared to the corresponding dsRNA in which the corresponding 2′position contains a hydrogen or an hydroxyl group. In yet otherembodiments, the dsRNA includes one or more linkages between adjacentnucleotides other than a naturally-occurring phosphodiester linkage.Examples of such linkages include phosphoramide, phosphorothioate, andphosphorodithioate linkages. The dsRNAs may also be chemically modifiednucleic acid molecules as taught in U.S. Pat. No. 6,673,661. In otherembodiments, the dsRNA contains one or two capped strands, as disclosed,for example, by WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No.60/130,377, filed Apr. 21, 1999. In other embodiments, the dsRNAcontains coding sequence or non-coding sequence, for example, aregulatory sequence (e.g., a transcription factor binding site, apromoter, or a 5′ or 3′ untranslated region (UTR) of an mRNA).Additionally, the dsRNA can be any of the at least partially dsRNAmolecules disclosed in WO 00/63364, filed Apr. 19, 2000 (see, forexample, pages 8-22), as well as any of the dsRNA molecules described inU.S. Provisional Application 60/399,998 filed Jul. 31, 2002, andPCT/US2003/024028, filed 31 Jul. 2003; and U.S. Provisional Application60/419,532 filed Oct. 18, 2002, and PCT/US2003/033466, filed 20 Oct.2003, the teaching of which is hereby incorporated by reference. Any ofthe dsRNAs may be expressed in vitro or in vivo using the methodsdescribed herein or standard methods, such as those described in WO00/63364, filed Apr. 19, 2000 (see, for example, pages 16-22). In somepreferred embodiments, multiple anti-HBV and/or anti-HCV dsRNA effectormolecules of the invention are transcribed in a mammalian cell from oneor more expression constructs each comprising multiple polymerase IIIpromoter expression cassettes as described in more detail in U.S.60/603,622; U.S. 60/629,942; and PCT/US05/29976 filed 23 Aug. 2005;“Multiple Polymerase III Promoter Expression Constructs”; the teachingof which is incorporated by reference.

dsRNA “Hairpin” Constructs or dsRNA “Hairpin” Expression Vectors:Constructs encoding a unimolecular hairpin dsRNA are more desirable forsome applications than constructs encoding duplex dsRNA (i.e., dsRNAcomposed of one RNA molecule with a sense region and a separate RNAmolecule with an antisense region) because the single-stranded RNA withinverted repeat sequences more efficiently forms a dsRNA hairpinstructure. This greater efficiency is due in part to the occurrence oftranscriptional interference arising in vectors containing convergingpromoters that generate duplex dsRNA. Transcriptional interferenceresults in the incomplete synthesis of each RNA strand thereby reducingthe number of complete sense and antisense strands that can base-pairwith each other and form duplexes. Transcriptional interference can beovercome, if desired, through the use of (i) a two vector system inwhich one vector encodes the sense RNA and the second vector encodes theantisense RNA, (ii) a bicistronic vector in which the individual strandsare encoded by the same plasmid but through the use of separatecistrons, or (iii) a single promoter vector that encodes a hairpindsRNA, i.e., an RNA in which the sense and antisense sequences areencoded within the same RNA molecule. Hairpin-expressing vectors havesome advantages relative to the duplex vectors. For example, in vectorsthat encode a duplex RNA, the RNA strands need to find and base-pairwith their complementary counterparts soon after transcription. If thishybridization does not happen, the individual RNA strands diffuse awayfrom the transcription template and the local concentration of sensestrands with respect to antisense strands is decreased. This effect isgreater for RNA that is transcribed intracellularly compared to RNAtranscribed in vitro due to the lower levels of template per cell.Moreover, RNA folds by nearest neighbor rules, resulting in RNAmolecules that are folded co-transcriptionally (i.e., folded as they aretranscribed). Some percentage of completed RNA transcripts is thereforeunavailable for base-pairing with a complementary second RNA because ofintra-molecular base-pairing in these molecules. The percentage of suchunavailable molecules increases with time following their transcription.These molecules may never form a duplex because they are already in astably folded structure. In a hairpin RNA, an RNA sequence is always inclose physical proximity to its complementary RNA. Since RNA structureis not static, as the RNA transiently unfolds, its complementarysequence is immediately available and can participate in base-pairingbecause it is so close. Once formed, the hairpin structure is predictedto be more stable than the original non-hairpin structure. Especiallydesirable are, e.g., “forced” hairpin constructs, partial hairpinscapable of being extended by RNA-dependent RNA polymerase to form dsRNAhairpins, as taught in U.S. Ser. No. 60/399,998P, filed 31 Jul. 2002;and PCT/US2003/024028, “Double Stranded RNA Structures and Constructsand Methods for Generating and Using the Same,” filed 31 Jul. 2003; aswell as the “udderly” structured hairpins, hairpins with mismatchedregions, and multiepitope constructs as taught in U.S. Ser. No.60/419,532, filed 18 Oct. 2002, and PCT/US2003/033466, “Double-StrandedRNA Structures and Constructs, and Methods for Generating and Using theSame,” filed 20 Oct. 2003.

By “short dsRNA” is meant a dsRNA that has about 200, 100, 75, 50, 45,40, 35, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20 or 19 contiguousnucleotides in length that are in a double stranded conformation.Desirably, the short dsRNA comprises a double-stranded region of atleast 19 contiguous basepairs in length identical/complementary to atarget sequence to be inhibited. In desirable embodiments, the doublestranded region is between 19 to 50, 19 to 40, 19 to 30, 19 to 25, 20 to25, 21 to 23, 25 to 30, or 30 to 40 contiguous basepairs in length,inclusive. In some embodiments, the short dsRNA is between 30 to 50, 50to 100, 100 to 200, 200 to 300, 400 to 500, 500 to 700, 700 to 1000,1000 to 2000, or 2000 to 5000 nucleotides in length, inclusive and has adouble stranded region that is between 38 and 60 contiguous basepairs inlength, inclusive. In one embodiment, the short dsRNA is completelydouble stranded. In some embodiments, the short dsRNA is between 11 and30 nucleotides in length, and the entire dsRNA is double stranded. Inother embodiments, the short dsRNA has one or two single strandedregions. In some embodiments, the short dsRNA is a “shRNA” or“short-hairpin RNA” or “shRNA effector molecule” or “dsRNA hairpin”,meaning an RNA molecule of less than approximately 400 to 500nucleotides (nt) in length, preferably less than 100 to 200 nt inlength, in which at least one stretch of at least about 15 to 100nucleotides (preferably 17 to 50 nt; more preferably 19 to 29 nt) isbase paired with a complementary sequence located on the same RNAmolecule, and where said sequence and complementary sequence areseparated by an unpaired region of at least about 4 to 7 nucleotides(preferably about 9 to about 15 nucleotides) which forms asingle-stranded loop above the stem structure created by the two regionsof base complementarity. The shRNA molecules comprise at least onestem-loop structure comprising a double-stranded stem region of about 17to about 100 bp; about 17 to about 50 bp; about 40 to about 100 bp;about 18 to about 40 bp; or from about 19 to about 29 bp; homologous andcomplementary to a target sequence to be inhibited; and an unpaired loopregion of at least about 4 to 7 nucleotides; preferably about 9 to about15 nucleotides, which forms a single-stranded loop above the stemstructure created by the two regions of base complementarity. IncludedshRNAs are dual or bi-finger (i.e., having two stem-loop structures) andmulti-finger hairpin dsRNAs (having multiple stem-loop structures), inwhich the RNA molecule comprises two or more of such stem-loopstructures separated by single-stranded spacer regions. In someembodiments, an expression construct may be used to express one or moreof such shRNA molecules in a mammalian cell, including multiple copiesof the same, and/or one or more, including multiple different, shorthairpin RNA molecules. Short hairpin RNA molecules considered to be the“same” as each other are those that comprise only the samedouble-stranded sequence, and short hairpin RNA molecules considered tobe “different” from each other will comprise different double-strandedsequences, regardless of whether the sequences to be targeted by eachdifferent double-stranded sequence are within the same, or a differentgene, such as, e.g., sequences of a promoter region and of a transcribedregion (mRNA) of the same gene, or sequences of two different genes.

In particular embodiments, the short dsRNA binds PKR or another proteinin a dsRNA-mediated stress response pathway. Desirably, such a shortdsRNA inhibits the dimerization and activation of PKR by at least 20,40, 60, 80, 90, or 100%. In some desirable embodiments, the short dsRNAinhibits the binding of a long dsRNA to PKR or another component of adsRNA-mediated stress response pathway by at least 20, 40, 60, 80, 90,or 100%. See also the teaching of U.S. Ser. No. 10/425,006, filed 28Apr. 2003, “Methods of Silencing Genes Without Inducing Toxicity”,Pachuk, as to utilization of short dsRNAs in conjunction with otherdsRNAs to avoid dsRNA-mediated toxicity. The applicants havedemonstrated, however, that dsRNA molecules, even long dsRNA molecules,are in general unlikely to evoke a significant dsRNA stress response,including a PKR or interferon or “panic” response, if they are expressedintracellularly in the mammalian (or other vertebrate) cell in which theRNAi effect is desired. See, e.g., US 2002/0132257, “Use ofpost-transcriptional gene silencing for identifying nucleic acidsequences that modulate the function of a cell”. Accordingly, such“expressed interfering RNA molecules” or “eiRNA” molecules and “eiRNAexpression constructs”, i.e., dsRNA molecules (or the correspondingdsRNA expression constructs) expressed intracellularly or endogenouslyin vivo within the mammalian cell in which dsRNA gene silencing or RNAiis induced, are preferred in some aspects of the invention.

By “at least 19 contiguous base pair nucleotide sequence” is meant thata nucleotide sequence can start at any nucleotide within one of thedisclosed sequences, so long as the start site is capable of producing apolynucleotide of at least 19 contiguous base pairs. For example, an atleast 19 contiguous base pair nucleotide sequence can comprisenucleotide 1 through nucleotide 19, nucleotide 2 through nucleotide 20,nucleotide 3 through nucleotide 21, and so forth to produce a 19mer.Thus, a 20mer can comprise nucleotide 1 through nucleotide 20,nucleotide 2 through nucleotide 21, nucleotide 3 through nucleotide 22,and so forth. Similar sequences above 20 contiguous nucleotides, e.g.,21, 22, 23, 24, 25, 26, 27, etc. selected from within the conservedsequences are envisioned. Such a sequence of at least 19 contiguousnucleotides (in double-stranded conformation with its complement) is “anat least 19 contiguous base pair sequence” and may be present as aduplex dsRNA, within a dsRNA hairpin, or encoded in a dsRNA expressionconstruct.

By “expression vector” is meant any double stranded DNA or doublestranded RNA designed to transcribe an RNA, e.g., a construct thatcontains at least one promoter operably linked to a downstream gene orcoding region of interest (e.g., a cDNA or genomic DNA fragment thatencodes a protein, or any RNA of interest, optionally, e.g., operativelylinked to sequence lying outside a coding region, an antisense RNAcoding region, a dsRNA coding region, or RNA sequences lying outside acoding region). Transfection or transformation of the expression vectorinto a recipient cell allows the cell to express RNA or protein encodedby the expression vector. An expression vector may be a geneticallyengineered plasmid, virus, or artificial chromosome derived from, forexample, a bacteriophage, adenovirus, retrovirus, poxvirus, orherpesvirus.

By an “expression construct” is meant any double-stranded DNA ordouble-stranded RNA designed to transcribe an RNA, e.g., a constructthat contains at least one promoter operably linked to a downstream geneor coding region of interest (e.g., a cDNA or genomic DNA fragment thatencodes a protein, or any RNA of interest). Transfection ortransformation of the expression construct into a recipient cell allowsthe cell to express RNA or protein encoded by the expression construct.An expression construct may be a genetically engineered plasmid, virus,or artificial chromosome derived from, for example, a bacteriophage,adenovirus, retrovirus, poxvirus, or herpesvirus. An expressionconstruct does not have to be replicable in a living cell, but may bemade synthetically. An expression construct or expression vectorengineered to express a double-stranded RNA effector molecule or dsRNAmolecule is a “dsRNA expression construct” or “dsRNA expression vector”.

In one embodiment of the invention, a recombinant expression vector orexpression construct is engineered to express multiple, e.g., three,four, five or more short hairpin dsRNA effector molecules, eachexpressed from a different expression cassette comprising a polymeraseIII promoter, one or more, including all of which, may be different fromthe others. In one aspect of the invention, a recombinant expressionvector transcribing three, four, five or more different shRNA molecules(each comprising a double-stranded “stem” region comprising at least 19contiguous basepairs from/complementary to a conserved HBV and/or HCVsequence) is used to inhibit replication of hepatitis B virus (HBV)and/or hepatitis C virus (HCV). In one embodiment, each shRNA moleculeis expressed under the control of a polymerase III promoter, e.g., 7SK,H1, and U6, which may be the same of different. Such dsRNA expressionconstructs comprising multiple polymerase III expression cassettes aredescribed in greater detail in PCT/US05/29976, “Multiple Polymerase IIIPromoter Expression Constructs”, the teaching of which is herebyincorporated by reference. In one aspect, a recombinant expressionvector or expression construct of the invention may express one or morebi-fingered or multi-fingered dsRNA hairpin molecules from one or morepolymerase III promoter-driven transcription units as well as one ormore single hairpin dsRNA molecules from one or more polymerase IIIpromoter-driven transcription units. It will be understood that in anyof said expression constructs transcribing a hairpin dsRNA from apolymerase III promoter, the hairpin dsRNA may be a single hairpin dsRNAor a bi-fingered, or multi-fingered dsRNA hairpin as described inWO2004/035765, published 29 Apr. 2004, or a partial or forced hairpinstructure as described in WO2004/011624, published 5 Feb. 2004, theteaching of which is incorporated herein by reference.

By “operably linked” is meant that a nucleic acid sequence or moleculeand one or more regulatory sequences (e.g., a promoter, enhancer,repressor, terminator) are connected in such a way as to permittranscription of an RNA molecule, e.g., a single-stranded RNA moleculesuch as a sense, antisense, a dsRNA hairpin, or an mRNA, or permitexpression and translation and/or secretion of the product (i.e., apolypeptide) of the nucleic acid molecule when the appropriate moleculesare bound to the regulatory sequences.

By a “promoter” is meant a nucleic acid sequence sufficient to directtranscription of a covalently linked nucleic acid molecule. Alsoincluded in this definition are those transcription control elements(e.g., enhancers) that are sufficient to render promoter-dependent geneexpression controllable in a cell type-specific, tissue-specific, ortemporal-specific manner, or that are inducible by external signals oragents; such elements, which are well-known to skilled artisans, may befound in a 5′ or 3′ region of a gene or within an intron. See, e.g.,published U.S. Patent Application No. 2005/0130184 A1, 16 Jun. 2005, Xuet al., directed to modified polymerase III promoters which utilizepolymerase II enhancer elements, as well as Published U.S. PatentApplication No. 2005/0130919 A1, 16 Jun. 2005, Xu et al., directed toregulatable polymerase III and polymerase II promoters, the teaching ofwhich is hereby incorporated by reference. Desirably a promoter isoperably linked to a nucleic acid sequence, for example, a cDNA or agene sequence, or a sequence encoding a dsRNA, e.g., a shRNA, in such away as to permit expression of the nucleic acid sequence.

The RNA molecule according to this invention may be delivered to themammalian cell or extracellular pathogen present in the mammalian cellin the composition as a dsRNA effector molecule or partially doublestranded RNA sequence, or RNA/DNA hybrid, which was made in vitro byconventional enzymatic synthetic methods using, for example, thebacteriophage T7, T3 or SP6 RNA polymerases according to theconventional methods described by such texts as the Promega Protocolsand Applications Guide, (3rd ed. 1996), eds. Doyle, ISBN No. 1 57Alternatively these molecules may be made by chemical synthetic methodsin vitro [see, e.g., Q. Xu et al., Nucleic Acids Res., 24(18):3643-4(September 1996); N. Naryshkin et al., Bioorg. Khim., 22(9):691-8(September 1996); J. A. Grasby et al., Nucleic Acids Res.,21(19):4444-50 (September 1993); C. Chaix et al., Nucleic Acids Res.17:7381-93 (1989); S. H. Chou et al., Biochem., 28(6):2422-35 (March1989); 0. Odal el al., Nucleic Acids Symp. Ser., 21:105-6 (1989); N. A.Naryshkin et al., Bioorg. Khim, 22(9):691-8 (September 1996); S. Sun etal., RNA, 3(11):1352-1363 (November 1997); X. Zhang et al., NucleicAcids Res., 25(20):3980-3 (October 1997); S. M. Grvaznov el al., NucleicAcids Res., 2-6 (18):4160-7 (September 1998); M. Kadokura et al.,Nucleic Acids Symp. Ser., 37:77-8 (1997); A. Davison et al., Biorned.Pept. Proteins. Nucleic Acids, 2(I):1-6 (1996); and A. V. Mudrakovskaiaet al., Bioorg. Khirn., 17(6):819-22 (June 1991)].

Still alternatively, the RNA molecule of this invention can be made in arecombinant microorganism, e.g., bacteria and yeast or in a recombinanthost cell, e.g., mammalian cells, and isolated from the cultures thereofby conventional techniques. See, e.g., the techniques described inSambrook et al, MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.; ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, which isexemplary of laboratory manuals that detail these techniques, and thetechniques described in U.S. Pat. Nos. 5,824,538; 5,877,159; and5,643,771, incorporated herein by reference.

Such RNA molecules prepared or synthesized in vitro may be directlydelivered to the mammalian cell or to the mammal as they are made invitro. The references above provide one of skill in the art with thetechniques necessary to produce any of the following specificembodiments, given the teachings provided herein. Therefore, in oneembodiment, the “agent” of the composition is a duplex (i.e., it is madeup of two strands), either complete or partially double stranded RNA.

In another embodiment, the agent is a single stranded RNA sense strand.In another embodiment, the agent of the composition is a single strandedRNA anti-sense strand.

Preferably the single stranded RNA sense or anti-sense strand forms ahairpin at one or both termini. Desirably, the single stranded RNA senseor anti-sense strand forms a hairpin at some intermediate portionbetween the termini. Such a single stranded RNA sense or anti-sensestrand may also be designed to fold back upon itself to become partiallydouble stranded in vitro or in vivo. Yet another embodiment of an extantRNA molecule as the effective agent used in the compositions is a singlestranded RNA sequence comprising both a sense polynucleotide sequenceand an antisense polynucleotide sequence, optionally separated by anon-base paired polynucleotide sequence. Preferably, this singlestranded RNA sequence has the ability to become double-stranded once itis in the cell, or in vitro during its synthesis. In desirableembodiments, a sequence of at least about 19 to 29 contiguous basepairswill assume a double-stranded conformation. In desirable embodiments,the double-stranded region will include an at least about 19 contiguousbasepair sequence identical/complementary to a target nucleotidesequence to be downregulated or inhibited.

Still another embodiment of this invention is an RNA/DNA hybrid asdescribed above.

Still another embodiment of the synthetic RNA molecule is a circular RNAmolecule that optionally forms a rod structure [see, e.g., K-S. Wang etal., Nature 323:508-514 (1986)] or is partially double-stranded, and canbe prepared according to the techniques described in S. Wang et al.,Nucleic Acids Res., 22(12):2326-33 (June 1994); Y. Matsumoto et al.,Proc. Natl. Acad. Sci, USA, 87(19):7628-32 (October 1990); E. Ford & M.Ares, Proc. Natl. Acad. Sci. USA 91(8):3117-21 (April 1994); M. Tsagriset al., Nucleic Acids Res., 19 7):1605-12 (April 1991); S. Braun et al.,Nucleic Acids Res. 24(21):4152-7 (November 1996); Z. Pasman et al., RNA,2(6):603-10 (June 1996); P. G. Zaphiropoulos, Proc. Natl. Acad. Sci.,USA, 93(13):6536-41 (June 1996); D. Beaudry et al., Nucleic Acids Res.,23(15):3064-6 (August 1995), all incorporated herein by reference. Stillanother agent is a double-stranded molecule comprised of RNA and DNApresent on separate strands, or interspersed on the same strand.

Alternatively, the RNA molecule may be formed in vivo and thus deliveredby a “delivery agent” which generates such a partially double-strandedRNA molecule in vivo after delivery of the agent to the mammalian cellor to the mammal. Thus, the agent which forms the composition of thisinvention is, in one embodiment, a double stranded DNA molecule“encoding” one of the above-described RNA molecules, e.g., a dsRNAexpression vector or expression construct. The DNA agent provides thenucleotide sequence which is transcribed within the cell to become adouble stranded RNA. In another embodiment, the DNA sequence provides adeoxyribonucleotide sequence which within the cell is transcribed intothe above-described single stranded RNA sense or anti-sense strand,which optionally forms a hairpin at one or both termini or folds backupon itself to become partially double stranded. The DNA molecule whichis the delivery agent of the composition can provide a single strandedRNA sequence comprising both a sense polynucleotide sequence and ananti-sense polynucleotide sequence, optionally separated by a nonbasepaired polynucleotide sequence, and wherein the single stranded RNAsequence has the ability to become double-stranded. Alternatively, theDNA molecule which is the delivery agent provides for the transcriptionof the above-described circular RNA molecule that optionally forms a rodstructure or partial double strand in vivo. The DNA molecule may alsoprovide for the in vivo production of an RNA/DNA hybrid as describedabove, or a duplex containing one RNA strand and one DNA strand. Thesevarious DNA molecules may be designed by resort to conventionaltechniques such as those described in Sambrook, cited above or in thePromega reference, cited above.

A latter delivery agent of the present invention, which enables theformation in the mammalian cell of any of the above-described RNAmolecules, can be a DNA single stranded or double stranded plasmid orvector. Expression vectors designed to produce RNAs as described hereinin vitro or in vivo may contain sequences under the control of any RNApolymerase, including mitochondria! RNA polymerase, RNA pol I, RNA polII, and RNA pol III, and viral polymerases, and bacteriophagepolymerases such as T7 and Sp6. Desirably, expression vectors designedfor in vivo expression of dsRNA effector molecules within a mammaliancell may be designed to utilize an endogenous mammalian polymerase suchas an RNA polymerase I, RNA polymerase II, RNA polymerase III, and/or amitochondrial polymerase. Expression vectors utilizing cognatepromoter(s), e.g., polymerase III promoters such as U6, H1, or 7SK, inorder to effect transcription by RNA polymerase III can readily bedesigned. Preferred for expression of short RNA molecules less thanabout 400 to 500 nucleotides in length are RNA polymerase III promoters.In some aspects, an “RNA polymerase III promoter” or “RNA pol IIIpromoter” or “polymerase III promoter” or “pol III promoter” ispreferred, meaning any invertebrate, vertebrate, or mammalian promoter,e.g., human, murine, porcine, bovine, primate, simian, etc. that, in itsnative context in a cell, associates or interacts with RNA polymeraseIII to transcribe its operably linked gene, or any variant thereof,natural or engineered, that will interact in a selected host cell withan RNA polymerase III to transcribe an operably linked nucleic acidsequence. Preferred in some applications are the Type III RNA pol IIIpromoters including U6, H1, 7SK, and MRP which exist in the 5′ flankingregion, include TATA boxes, and lack internal promoter sequences. Onereason RNA Pol III promoters are especially desirable for expression ofsmall engineered RNA transcripts is that RNA Pol III termination, unlikeRNA polymerase II termination, occurs efficiently and precisely at ashort run of thymine residues in the DNA coding strand, without otherprotein factors, T4 and T5 being the shortest Pol III terminationsignals in yeast and mammals, with oligo (dT) terminators longer than T5being very rare in mammals. Accordingly, the multiple polymerase IIIpromoter expression constructs of the invention will include anappropriate oligo (dT) termination signal, i.e., a sequence of 4, 5, 6or more Ts, operably linked 3′ to each RNA Pol III promoter in the DNAcoding strand.

These vectors can be used to transcribe the desired RNA molecule in thecell according to this invention. Vectors may be desirably designed toutilize an endogenous mitochondrial RNA polymerase (e.g., humanmitochondrial RNA polymerase, in which case such vectors may utilize thecorresponding human mitochondrial promoter). Mitochondria! polymerasesmay be used to generate capped (through expression of a capping enzyme)or uncapped messages in vivo. RNA pol I, RNA pol II, and RNA pol IIItranscripts may also be generated in vivo. Such RNAs may be capped ornot, and if desired, cytoplasmic capping may be accomplished by variousmeans including use of a capping enzyme such as a vaccinia cappingenzyme or an alphavirus capping enzyme. However, all pol II transcriptsare capped. The DNA vector is designed to contain one of the promotersor multiple promoters in combination (mitochondrial, RNA pol I, pol II,or pol III, or viral, bacterial or bacteriophage promoters along withthe cognate polymerases). Preferably, where the promoter is RNA pol II,the sequence encoding the RNA molecule has an open reading frame greaterthan about 300 nts and must follow the rules of design to preventnonsense-mediated degradation in the nucleus. Such plasmids or vectorscan include plasmid sequences from bacteria, viruses or phages.

Such vectors include chromosomal, episomal and virus-derived vectors,e.g., vectors derived from bacterial plasmids, bacteriophages, yeastepisomes, yeast chromosomal elements, and viruses, vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, cosmids and phagemids.

Thus, one exemplary vector is a single or double-stranded phage vector.Another exemplary vector is a single or double-stranded RNA or DNA viralvector. Such vectors may be introduced into cells as polynucleotides,preferably DNA, by well known techniques for introducing DNA and RNAinto cells. The vectors, in the case of phage and viral vectors may alsobe and preferably are introduced into cells as packaged or encapsidatedvirus by well known techniques for infection and transduction. Viralvectors may be replication competent or replication defective. In thelatter case, viral propagation generally occurs only in complementinghost cells.

In another embodiment the delivery agent comprises more than a singleDNA or RNA plasmid or vector. As one example, a first DNA plasmid canprovide a single stranded RNA sense polynucleotide sequence as describedabove, and a second DNA plasmid can provide a single stranded RNAanti-sense polynucleotide sequence as described above, wherein the senseand anti-sense RNA sequences have the ability to base-pair and becomedouble-stranded. Such plasmid(s) can comprise other conventional plasmidsequences, e.g., bacterial sequences such as the well-known sequencesused to construct plasmids and vectors for recombinant expression of aprotein. However, it is desirable that the sequences which enableprotein expression, e.g., Kozak regions, etc., are not included in theseplasmid structures.

The vectors designed to produce dsRNAs of the invention may desirably bedesigned to generate two or more, including a number of different dsRNAshomologous and complementary to a target sequence. This approach isdesirable in that a single vector may produce many, independentlyoperative dsRNAs rather than a single dsRNA molecule from a singletranscription unit and by producing a multiplicity of different dsRNAs,it is possible to self select for optimum effectiveness. Various meansmay be employed to achieve this, including autocatalytic sequences aswell as sequences for cleavage to create random and/or predeterminedsplice sites.

Other delivery agents for providing the information necessary forformation of the above-described desired RNA molecules in the mammaliancell include live, attenuated or killed, inactivated recombinantbacteria which are designed to contain the sequences necessary for therequired RNA molecules of this invention. Such recombinant bacterialcells, fungal cells and the like can be prepared by using conventionaltechniques such as described in U.S. Pat. Nos. 5,824,538; 5,877,159; and5,643,771, incorporated herein by reference. Microorganisms useful inpreparing these delivery agents include those listed in the above citedreference, including, without limitation, Escherichia coli, Bacillussubtilis, Salmonella typhimurium, and various species of Pseudomonas,Streptomyces, and Staphylococcus.

Still other delivery agents for providing the information necessary forformation of the desired, above-described RNA molecules in the mammaliancell include live, attenuated or killed, inactivated viruses, andparticularly recombinant viruses carrying the required RNApolynucleotide sequence discussed above. Such viruses may be designedsimilarly to recombinant viruses presently used to deliver genes tocells for gene therapy and the like, but preferably do not have theability to express a protein or functional fragment of a protein. Amonguseful viruses or viral sequences which may be manipulated to providethe required RNA molecule to the mammalian cell in vivo are, withoutlimitation, alphavirus, adenovirus, adeno associated virus,baculoviruses, delta virus, pox viruses, hepatitis viruses, herpesviruses, papova viruses (such as SV40), poliovirus, pseudorabiesviruses, retroviruses, lentiviruses, vaccinia viruses, positive andnegative stranded RNA viruses, viroids, and virusoids, or portionsthereof. These various viral delivery agents may be designed by applyingconventional techniques such as described in M. Di Nocola et al., CancerGene Ther., 5(6):350-6 (1998), among others, with the teachings of thepresent invention.

The term “in vivo” is intended to include any system wherein thecellular DNA or RNA replication machinery is intact, preferably withinintact living cells, including tissue culture systems, tissue explants,and within single cell or multicellular living organisms.

By “multiple sequitope dsRNA” or “multisequitope dsRNA” or “multipleepitope dsRNA” is meant an RNA molecule that has segments derived frommultiple target nucleic acids or that has non-contiguous segments fromthe same target nucleic acid. For example, the multiple sequitope dsRNAmay have segments derived from (i) sequences representing multiple genesof a single organism; (ii) sequences representing one or more genes froma variety of different organisms; and/or (iii) sequences representingdifferent regions of a particular gene (e.g., one or more sequences froma promoter and one or more sequences from an mRNA. Desirably, eachsegment has substantial sequence identity to the corresponding region ofa target nucleic acid. In various desirable embodiments, a segment withsubstantial sequence identity to the target nucleic acid is at least 19,20, 21, 22, 23, 24, 25, 26, 27, 30, 40, 50, 100, 200, 500, 750, or morebasepairs in length. In desirable embodiments, the multiple epitopedsRNA inhibits the expression of at least 2, 4, 6, 8, 10, 15, 20, ormore target genes by at least 20, 40, 60, 80, 90, 95, or 100%. In someembodiments, the multiple epitope dsRNA has non-contiguous segments fromthe same target gene or from the same target polynucleotide that may ormay not be in the naturally occurring 5′ to 3′ order of the segments,and the dsRNA inhibits the expression of the target nucleic acid by atleast 50, 100, 200, 500, or 1000% more than a dsRNA with only one of thesegments.

By “sequitope” is meant a contiguous sequence of double-strandedpolyribonucleotides that can associate with and activate RISC(RNA-induced silencing complex), usually a contiguous sequence ofbetween 19 and 27 basepairs, inclusive. Sequences comprising at leastone sequitope from within one or more of the conserved HBV and/or HCVnucleotide sequences identified here may be utilized for dsRNA mediatedgene silencing as taught herein.

Multiple-epitope/multiple-sequitope dsRNAs the advantages of amultiple-epitope or multisequitope double-stranded RNA approach astaught in U.S. Ser. No. 60/419,532, filed 18 Oct. 2002 andPCT/US2003/033466, filed 20 Oct. 2003, are applicable to utilization ofthe conserved HBV and/or HCV sequences of the invention. Because asingular species of dsRNA can simultaneously silence many target genes(e.g., genes from multiple pathogens, multiple genes or sequences from asingle pathogen, or genes associated with multiple diseases), a multipleepitope dsRNA can be used for many different indications in the samesubject or used for a subset of indications in one subject and anothersubset of indications in another subject. For such applications, theability to express long dsRNA molecules (e.g., dsRNA molecules withsequences from multiple genes) without invoking the dsRNA stressresponse is highly desirable. For example, by using a series ofsequences, each, e.g., as short as 19-21 nucleotides, desirably 100 to600 nucleotides, or easily up to 1, 2, 3, 4, 5, or more kilobases suchthat the total length of such sequences is within the maximum capacityof the selected plasmid (e.g., 20 kilobases in length), a single suchpharmaceutical composition can provide protection against a large numberof pathogens and/or toxins at a relatively low cost and low toxicity,e.g., HBV, HCV, HIV, etc.

The use of multiple epitopes or sequitopes derived from one or moregenes and/or different overlapping and/or non-contiguous sequences ofthe same polynucleotide or gene from multiple strains and/or variants ofa highly variable or rapidly mutating pathogen such as HBV and/or HCVcan also be very advantageous. For example, a singular dsRNA speciesthat recognizes and targets multiple strains and/or variants of HBVand/or HCV can be used as a universal treatment or vaccine for thevarious strains/variants of HBV and/or HCV.

The ability to silence multiple genes of a particular pathogen such asHBV and/or HCV prevents the selection of, in this case, HBV and/or HCV“escape mutants.” In contrast, typical small molecule treatment orvaccine therapy that only targets one gene or protein results in theselection of pathogens that have sustained mutations in the target geneor protein and the pathogen thus becomes resistant to the therapy. Bysimultaneously targeting a number of genes or sequences of the pathogenand/or extensive regions of the pathogen using the multiple epitopeapproach of the present invention, the emergence of such “escapemutants” is effectively precluded.

For example, it is considered particularly advantageous to include amixture of sequences from both HCV SEQ ID NO:11 and SEQ ID NO:12, andSEQ ID NO: 27, i.e., one or more sequences (e.g, each at least 19, 20,21, 22, 23, 24, 25, 26, 27 to 29 contiguous nucleotides) from HCV SEQ IDNO:11 together with one or more sequences (e.g, each at least 19, 20,21, 22, 23, 24, 25, 26, 27 to 29 contiguous nucleotides) from HCV SEQ IDNO:12 and from SEQ ID NO: 27, either in a single dsRNA molecule, anadmixture of dsRNA molecules, or through concomitant administration ofsuch molecules to a patient (or by administering one or more dsRNAexpression constructs which produce such dsRNA moleculesintracellularly), in order to decrease the ability of the virus togenerate viable escape mutants. Similarly, it would be advantageous toprovide a mixture of dsRNA molecules comprising a number of theconserved HBV sequences, in some cases in combination with one or moreof the conserved HCV sequences of the invention.

Similarly, it may be desirable to use sequences from two or more of HBVSEQ ID NO:1, SEQ ID NO:2, AND SEQ ID NO:3, either in a single dsRNAconstruct, an admixture of constructs, or through concomitantadministration of such constructs (or dsRNA expression constructs whichproduce such dsRNA molecules) to a patient. SEQ ID NO:1, SEQ ID NO:2,and SEQ ID NO:3 map to the HBV surface antigen genes. Due to theoverlapping nature of the HBV mRNAs, the following mRNAs would betargeted by one of more of these sequences: Surface Ag (sAg) mRNAs,precore, core and polymerase mRNAs. However, since sAg mRNAs are themost abundant, it is more likely that these mRNAs will be targeted ifthe gene-silencing machinery is saturable. It is possible, however, thatall listed mRNAs will be targeted. Reduction of surface Ag is desirablefor several reasons: a) surface Ag is needed for assembly of infectiousvirions; b) overexpression of Surface Ag during infection is thought tocontribute to immune anergy that occurs during chronic HBV infection;and c) the expression of HBVsAg in the livers of infected individuals(even in the absence of virus, i.e., from integrated sAg sequences intothe host genome) induces hepatitis. Therefore, reduction of sAg islikely to decrease viral titers, overcome immune anergy anddecrease/prevent hepatitis.

HBV SEQ ID NO:4 maps to the unique region of precore and core and willtarget these mRNAs specifically. Core protein is needed to makefunctional virions and so down regulation of this mRNA is predicted todecrease viral titers. There should be no competition of these effectorRNAs for surface, polymerase or X mRNAs.

HBV SEQ ID NO:5 through SEQ ID NO:8 map to the polymerase gene. EffectorRNAs are predicted to target only precore/core and polymerasetranscripts. There should be no competition with sAg or X mRNAs.Polymerase is needed for the synthesis of viral genomes and thereforeviral particle titer is expected to decrease as polymerase is decreased.

HBV SEQ ID NO:9 maps to the X gene. Due to the terminal redundancy ofall the HBV mRNAs, these effector RNAs have the potential to target allof the HBV viral mRNAs. X protein has many ascribed (non proven)functions. Evidence is emerging, however, that X-gene expression isassociated with hepatocellular carcinogenesis, in part related topromotion of detachment and migration of cells out of the primary tumorsite. Since the X gene is often found in integrated HBV sequences inindividuals with and without active hepatitis, down-regulation of X geneexpression is predicted to ameliorate disease, including the incidenceof hepatocellular carcinoma.

In general, the more sequences or sequitopes from the differentidentified sequences that are used (e.g., from SEQ ID NO:1, SEQ ID NO:2,and/or SEQ ID NO:3, plus sequences from SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10), the lesslikely a virus will be able to generate viable escape mutants. Also, themore different mRNAs that can be targeted, the more significant will bethe drops in viral titer and disease amelioration.

Desirable combinations for multiepitope or multisequitope dsRNAexpression constructs or dsRNA effector molecules, an admixture of dsRNAexpression constructs or dsRNA effector molecules, or the concomitantadministration of different dsRNA expression constructs or dsRNAeffector molecules include the following: Sequences from SEQ ID NO:1,SEQ ID NO:2, or SEQ ID NO:3 plus sequences from SEQ ID NO:4; Sequencesfrom SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 plus sequences from SEQ IDNO:5; Sequences from SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 plussequences from SEQ ID NO:6; Sequences from SEQ ID NO:1, SEQ ID NO:2, orSEQ ID NO:3 plus sequences from SEQ ID NO:7; Sequences from SEQ ID NO:1,SEQ ID NO:2, or SEQ ID NO:3 plus sequences from SEQ ID NO:8; Sequencesfrom SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 plus sequences from SEQ IDNO:9; Sequences from SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 plussequences from SEQ ID NO:10; Sequences from SEQ ID NO:1, SEQ ID NO:2, orSEQ ID NO:3 plus sequences from SEQ ID NO:4 and SEQ ID NO:5; Sequencesfrom SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 plus sequences from SEQ IDNO:4 and SEQ ID NO:6; Sequences from SEQ ID NO:1, SEQ ID NO:2, or SEQ IDNO:3 plus sequences from SEQ ID NO:4 and SEQ ID NO:7; Sequences from SEQID NO:1, SEQ ID NO:2, or SEQ ID NO:3 plus sequences from SEQ ID NO:4 andSEQ ID NO:8; Sequences from SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3plus sequences from SEQ ID NO:4 and SEQ ID NO:9; Sequences from SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3 plus sequences from SEQ ID NO:4 andSEQ ID NO:10; Sequences from SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3plus sequences from SEQ ID NO:5 and SEQ ID NO:6; Sequences from SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3 plus sequences from SEQ ID NO:5 andSEQ ID NO:7; Sequences from SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3plus sequences from SEQ ID NO:5 and SEQ ID NO:8; Sequences from SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3 plus sequences from SEQ ID NO:5 andSEQ ID NO:9; Sequences from SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3plus sequences from SEQ ID NO:5 and SEQ ID NO:10; Sequences from SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3 plus sequences from SEQ ID NO:6 andSEQ ID NO:7; Sequences from SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3plus sequences from SEQ ID NO:6 and SEQ ID NO:8; Sequences from SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3 plus sequences from SEQ ID NO:6 andSEQ ID NO:9; Sequences from SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3plus sequences from SEQ ID NO:6 and SEQ ID NO:10; Sequences from SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3 plus sequences from SEQ ID NO:7 andSEQ ID NO:8; Sequences from SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3plus sequences from SEQ ID NO:7 and SEQ ID NO:9; Sequences from SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3 plus sequences from SEQ ID NO:7 andSEQ ID NO:10; Sequences from SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3plus sequences from SEQ ID NO:8 and SEQ ID NO:9; Sequences from SEQ IDNO:1, SEQ ID NO:2, or SEQ ID NO:3 plus sequences from SEQ ID NO:8 andSEQ ID NO:10; Sequences from SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3plus sequences from SEQ ID NO:9 and SEQ ID NO:10; Sequences from SEQ IDNO:4 and SEQ ID NO:5; Sequences from SEQ ID NO:4 and SEQ ID NO:6;Sequences from SEQ ID NO:4 and SEQ ID NO:7; Sequences from SEQ ID NO:4and SEQ ID NO:8; Sequences from SEQ ID NO:4 and SEQ ID NO:9; Sequencesfrom SEQ ID NO:4 and SEQ ID NO:10; Sequences from SEQ ID NO:5 and SEQ IDNO:6; Sequences from SEQ ID NO:5 and SEQ ID NO:7; Sequences from SEQ IDNO:5 and SEQ ID NO:8; Sequences from SEQ ID NO:5 and SEQ ID NO:9;Sequences from SEQ ID NO:5 and SEQ ID NO:10; Sequences from SEQ ID NO:6and SEQ ID NO:7; Sequences from SEQ ID NO:6 and SEQ ID NO:8; Sequencesfrom SEQ ID NO:6 and SEQ ID NO:9; Sequences from SEQ ID NO:6 and SEQ IDNO:10; Sequences from SEQ ID NO:7 and SEQ ID NO:8; Sequences from SEQ IDNO:7 and SEQ ID NO:9; Sequences from SEQ ID NO:7 and SEQ ID NO:10;Sequences from SEQ ID NO:8 and SEQ ID NO:9; Sequences from SEQ ID NO:8and SEQ ID NO:10; Sequences from SEQ ID NO:9 and SEQ ID NO:10; Sequencesfrom SEQ ID NO:4, SEQ ID NO:5; and SEQ ID NO:6; Sequences from SEQ IDNO:4, SEQ ID NO:5; and SEQ ID NO:7; Sequences from SEQ ID NO:4, SEQ IDNO:5; and SEQ ID NO:8; Sequences from SEQ ID NO:4, SEQ ID NO:5; and SEQID NO:9; Sequences from SEQ ID NO:4, SEQ ID NO:5; and SEQ ID NO:10;Sequences from SEQ ID NO:4, SEQ ID NO:6; and SEQ ID NO:7; Sequences fromSEQ ID NO:4, SEQ ID NO:6; and SEQ ID NO:8; Sequences from SEQ ID NO:4,SEQ ID NO:6; and SEQ ID NO:9; Sequences from SEQ ID NO:4, SEQ ID NO:6;and SEQ ID NO:10; Sequences from SEQ ID NO:4, SEQ ID NO:7; and SEQ IDNO:8; Sequences from SEQ ID NO:4, SEQ ID NO:7; and SEQ ID NO:9;Sequences from SEQ ID NO:4, SEQ ID NO:7; and SEQ ID NO:10; Sequencesfrom SEQ ID NO:4, SEQ ID NO:8; and SEQ ID NO:9; Sequences from SEQ IDNO:4, SEQ ID NO:8; and SEQ ID NO:10; Sequences from SEQ ID NO:4, SEQ IDNO:9; and SEQ ID NO:10; Sequences from SEQ ID NO:5, SEQ ID NO:6; and SEQID NO:7; Sequences from SEQ ID NO:5, SEQ ID NO:6; and SEQ ID NO:8;Sequences from SEQ ID NO:5, SEQ ID NO:6; and SEQ ID NO:9; Sequences fromSEQ ID NO:5, SEQ ID NO:6; and SEQ ID NO:10; Sequences from SEQ ID NO:5,SEQ ID NO:7; and SEQ ID NO:8; Sequences from SEQ ID NO:5, SEQ ID NO:7;and SEQ ID NO:9; Sequences from SEQ ID NO:5, SEQ ID NO:7; and SEQ IDNO:10; Sequences from SEQ ID NO:5, SEQ ID NO:8; and SEQ ID NO:9;Sequences from SEQ ID NO:5, SEQ ID NO:8; and SEQ ID NO:10; Sequencesfrom SEQ ID NO:5, SEQ ID NO:9; and SEQ ID NO:10; Sequences from SEQ IDNO:6, SEQ ID NO:7; and SEQ ID NO:8; Sequences from SEQ ID NO:6, SEQ IDNO:7; and SEQ ID NO:9; Sequences from SEQ ID NO:6, SEQ ID NO:7; and SEQID NO:10; Sequences from SEQ ID NO:6, SEQ ID NO:8; and SEQ ID NO:9;Sequences from SEQ ID NO:6, SEQ ID NO:8; and SEQ ID NO:10; Sequencesfrom SEQ ID NO:6, SEQ ID NO:9; and SEQ ID NO:10; Sequences from SEQ IDNO:7, SEQ ID NO:8; and SEQ ID NO:9; Sequences from SEQ ID NO:7, SEQ IDNO:8; and SEQ ID NO:10; Sequences from SEQ ID NO:7, SEQ ID NO:9; and SEQID NO:10; Sequences from SEQ ID NO:8, SEQ ID NO:9; and SEQ ID NO:10;Sequences from SEQ ID NO:4, SEQ ID NO:5; SEQ ID NO:6; and SEQ ID NO:7;Sequences from SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:8;Sequences from SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:9;Sequences from SEQ ID NO:4, SEQ ID NO:5; SEQ ID NO:6; and SEQ ID NO:10;Sequences from SEQ ID NO:4, SEQ ID NO:5; SEQ ID NO:7; and SEQ ID NO:8;Sequences from SEQ ID NO:4, SEQ ID NO:5; SEQ ID NO:7; and SEQ ID NO:9;Sequences from SEQ ID NO:4, SEQ ID NO:5; SEQ ID NO:7; and SEQ ID NO:10;Sequences from SEQ ID NO:4, SEQ ID NO:5; SEQ ID NO:8; and SEQ ID NO:9;Sequences from SEQ ID NO:4, SEQ ID NO:5; SEQ ID NO:8; and SEQ ID NO:10;Sequences from SEQ ID NO:4, SEQ ID NO:5; SEQ ID NO:9; and SEQ ID NO:10;Sequences from SEQ ID NO:4, SEQ ID NO:6; SEQ ID NO:7; and SEQ ID NO:8;Sequences from SEQ ID NO:4, SEQ ID NO:6; SEQ ID NO:7; and SEQ ID NO:9;Sequences from SEQ ID NO:4, SEQ ID NO:6; SEQ ID NO:7; and SEQ ID NO:10;Sequences from SEQ ID NO:4, SEQ ID NO:7; SEQ ID NO:8; and SEQ ID NO:9;Sequences from SEQ ID NO:4, SEQ ID NO:7; SEQ ID NO:8; and SEQ ID NO:10;Sequences from SEQ ID NO:4, SEQ ID NO:7; SEQ ID NO:9; and SEQ ID NO:10;Sequences from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8;Sequences from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:9;Sequences from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:10;Sequences from SEQ ID NO:5, SEQ ID NO:6; SEQ ID NO:8; and SEQ ID NO:9;Sequences from SEQ ID NO:5, SEQ ID NO:6; SEQ ID NO:8; and SEQ ID NO:10;Sequences from SEQ ID NO:5, SEQ ID NO:6; SEQ ID NO:9; and SEQ ID NO:10;Sequences from SEQ ID NO:5, SEQ ID NO:7; SEQ ID NO:8; and SEQ ID NO:9;Sequences from SEQ ID NO:5, SEQ ID NO:7; SEQ ID NO:8; and SEQ ID NO:10;Sequences from SEQ ID NO:5, SEQ ID NO:7; SEQ ID NO:9; and SEQ ID NO:10;Sequences from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9;Sequences from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:10;Sequences from SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, and SEQ ID NO:10;Sequences from SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10;Sequences from SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, andSEQ ID NO:8; Sequences from SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQID NO:7, and SEQ ID NO:9; Sequences from SEQ ID NO:4, SEQ ID NO:5, SEQID NO:6, SEQ ID NO:7, and SEQ ID NO:10; Sequences from SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO:9; Sequences from SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, and SEQ ID NO:10;Sequences from SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:9, andSEQ ID NO:10; Sequences from SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQID NO:8, and SEQ ID NO:9; Sequences from SEQ ID NO:4, SEQ ID NO:5, SEQID NO:7, SEQ ID NO:8, and SEQ ID NO:10; Sequences from SEQ ID NO:4, SEQID NO:5, SEQ ID NO:7, SEQ ID NO:9, and SEQ ID NO:10; Sequences from SEQID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9;Sequences from SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, andSEQ ID NO:10; Sequences from SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQID NO:9, and SEQ ID NO:10; Sequences from SEQ ID NO:5, SEQ ID NO:6, SEQID NO:7, SEQ ID NO:8, and SEQ ID NO:9; Sequences from SEQ ID NO:5, SEQID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:10; Sequences from SEQID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:9, and SEQ ID NO:10;Sequences from SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, andSEQ ID NO:10; Sequences from SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQID NO:9, and SEQ ID NO:10; Sequences from SEQ ID NO:6, SEQ ID NO:7, SEQID NO:8, SEQ ID NO:9, and SEQ ID NO:10; Sequences from SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9;Sequences from SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQID NO:8, and SEQ ID NO:10; Sequences from SEQ ID NO:4, SEQ ID NO:5, SEQID NO:6, SEQ ID NO:7, SEQ ID NO:9, and SEQ ID NO:10; Sequences from SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, and SEQ IDNO:10; Sequences from SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:8, SEQ ID NO:9, and SEQ ID NO:10; Sequences from SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10; Sequencesfrom SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,and SEQ ID NO:10; and Sequences from SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10. Preferredin some aspects are sequences from SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, and SEQ ID NO:8, including combinations of sequitopes from SEQ IDNO:5, plus SEQ ID NO:6, plus SEQ ID NO:7, plus SEQ ID NO:8.

In another embodiment, combinations of sequitopes at least 19 contiguousbase pairs in length and longer sequences from within any of theaforementioned sequences (e.g., SEQ ID NO:1 through SEQ ID NO:12) may beutilized either in a single dsRNA expression construct or dsRNA effectormolecule, an admixture of dsRNA expression constructs or dsRNA effectormolecules or through concomitant administration of such dsRNA expressionconstructs or dsRNA effector molecules to a patient. By a sequence of“at least 19 contiguous base pairs in length” is meant that a sequenceor sequitope of at 19 contiguous bases in length is present indouble-stranded conformation, or within a double-stranded RNA effectormolecule.

As discussed elsewhere herein, a particularly preferred embodiment ofthe invention utilizes dsRNA expression constructs or vectors to achieveendogenous delivery of the dsRNAs of the invention, especially themultiple different sequences described above. These dsRNAs may beprovided e.g., on the same cistron of an expression construct such as aplasmid, on different cistrons of an expression construct, or ondifferent expression constructs or plasmids, e.g., one or more plasmidsand/or one or more vectors, including viral vectors. The combination ofdifferent dsRNA effector molecules such as shRNA effector molecules maybe provided to a mammalian cell by in vivo expression from a singleexpression construct such as a plasmid, with each dsRNA effectormolecule transcribed from a different expression cassette driven by adifferent promoter, e.g., an RNA polymerase I promoter and/or an RNApolymerase III promoter, e.g., a type 3 RNA polymerase III promoter suchas U6, H1, 7SK, or MRP. In some embodiments, each such differentexpression cassette may contain a different RNA polymerase III promoter,which may be the same or different, and an RNA polymerase IIItermination sequence. In another embodiment, a combination of differentdsRNA effector molecules such as shRNA effector molecules may beprovided to a mammalian cell by in vivo expression from a singleexpression construct such as a plasmid or a viral vector which comprisean expression cassette comprising multiple different promoters, e.g., anRNA polymerase I promoter and/or an RNA polymerase III promoter, e.g., atype 3 RNA polymerase III promoter such as U6, H1, 7SK, or MRP, andwherein each of such promoters transcribes a different dsRNA effectormolecule. Such multiple different dsRNA effector sequences may also beprovided to an in vivo mammalian cell exogenously, in any differentmixture of one or more dsRNA structures, duplexes and/or harpins, and/orin combination with one or more endogenously expressed dsRNA structures.

Desirable methods of administration of nucleic acids The DNA and/or RNAconstructs, e.g., dsRNA effector molecules, of the invention may beadministered to the host cell/tissue/organism as “naked” DNA, RNA, orDNA/RNA, formulated in a pharmaceutical vehicle without any transfectionpromoting agent. More efficient delivery may be achieved as known tothose of skill in the art of DNA and RNA delivery, using e.g., suchpolynucleotide transfection facilitating agents known to those of skillin the art of RNA and/or DNA delivery. The following are exemplaryagents: cationic amphiphiles including local anesthetics such asbupivacaine, cationic lipids, liposomes or lipidic particles,polycations such as polylysine, branched, three-dimensional polycationssuch as dendrimers, carbohydrates, detergents, or surfactants, includingbenzylammonium surfactants such as benzalkonium chloride. Non-exclusiveexamples of such facilitating agents or co-agents useful in thisinvention are described in U.S. Pat. Nos. 5,593,972; 5,703,055;5,739,118; 5,837,533; 5,962,482; 6,127,170; 6,379,965; and 6,482,804;and International Patent Application No. PCT/US98/22841; the teaching ofwhich is hereby incorporated by reference. U.S. Pat. Nos. 5,824,538;5,643,771; and 5,877,159 (incorporated herein by reference) teachdelivery of a composition other than a polynucleotide composition, e.g.,a transfected donor cell or a bacterium containing the dsRNA-encodingcompositions of the invention.

In some embodiments, the dsRNA or dsRNA expression vector is complexedwith one or more cationic lipids or cationic amphiphiles, such as thecompositions disclosed in U.S. Pat. No. 4,897,355 (Eppstein et al.,filed Oct. 29, 1987), U.S. Pat. No. 5,264,618 (Feigner et al., filedApr. 16, 1991) or U.S. Pat. No. 5,459,127 (Feigner et al., filed Sep.16, 1993). In other embodiments, the dsRNA or dsRNA expression vector iscomplexed with a liposome/liposomic composition that includes a cationiclipid and optionally includes another component such as a neutral lipid(see, for example, U.S. Pat. No. 5,279,833 (Rose), U.S. Pat. No.5,283,185 (Epand), and U.S. Pat. No. 5,932,241).

Particularly desirable methods and compositions for delivery of theoligonucleotide compositions of the invention for pharmaceuticalapplications, including for targeted delivery to hepatocytes, aredescribed in PCT/US03/14288, filed May 6, 2003, the teaching of which isincorporated herein by reference.

Transformation/transfection of the cell for research and othernon-therapeutic purposes may occur through a variety of means including,but not limited to, lipofection, DEAE-dextran-mediated transfection,microinjection, calcium phosphate precipitation, viral or retroviraldelivery, electroporation, or biolistic transformation. The RNA or RNAexpression vector (DNA) may be naked RNA or DNA or local anestheticcomplexed RNA or DNA (See U.S. Pat. Nos. 6,217,900 and 6,383,512,“Vesicular Complexes and Methods of Making and Using the Same, Pachuk etal., supra).

Another desirable delivery technology for the dsRNAs or dsRNA expressionconstructs of the invention for pharmaceutical applications is theself-assembling Cyclosert™ two-component nucleic acid delivery system,based on cyclodextrin-containing polycations, which are available fromInsert Therapeutics, Pasadena, Calif. (See Bioconjug Chem 2003 May-June;14 (3): 672-8; Popielarski et al.; “Structural effects ofcarbohydrate-containing polycations on gene delivery. 3. Cyclodextrintype and functionalization”; as well as Bioconjug Chem 2003January-February; 14 (1):247-54 and 255-61.) The first component is alinear, cyclodextrin-containing polycationic polymer, that when mixedwith DNA, binds to the phosphate “backbone” of the nucleic acid,condensing the DNA and self assembling into uniform, colloidalnanoparticles that protect the DNA from nuclease degradation in serum. Asecond component is a surface modifying agent with a terminaladamantine-PEG molecule, that when combined with the cyclodextrinpolymer forms an inclusion complex with surface cyclodextrins andprevents aggregation, enhances stability and enables systemicadministration. In addition, targeting ligands to cell surface receptorsmay be attached to the modifier providing for targeted delivery of DNAdirectly to target cells of interest. Since hepatocytes are susceptibleto HBV and HCV infection, utilizing this method to target delivery ofthe dsRNA expression constructs of the invention to liver cells isconsidered especially advantageous. E.g., the asialoglycoproteinreceptor (ASGP-R) on mammalian hepatocytes may be targeted by use ofsynthetic ligands with galactosylated or lactosylated residues, such asgalactosylated polymers.

In general, targeting for selective delivery of the dsRNA constructs ofthe invention to hepatocytes is preferred. Targeting to hepatocytes maybe achieved by coupling to ligands for hepatocyte-specific receptors.For example, asialo-orosomucoid, (poly)L-lysine-asialo-orosomucoid, orany other ligands of the hepatic asialoglycoprotein receptor (Spiess,Biochemistry 29(43):10009-10018, 1990; Wu et al., J. Biol. Chem.267(18):12436-12439, 1992; Wu et al., Biotherapy 3:87-95, 1991).Similarly, the oligonucleotides may be targeted to hepatocytes by beingconjugated to monoclonal antibodies that specifically bind tohepatocyte-specific receptors. Oligonucleotides may also be targeted tohepatocytes using specific vectors, as described below.

Particularly preferred compositions for delivery of dsRNAs or dsRNAexpression constructs of the invention are the multifunctionalcompositions as described in PCT/US03/14288, filed May 6, 2003, whichmay include trilactosyl spermine as a ligand for targeting to the ASGReceptor of hepatocytes. Trilactosyl cholesteryl spermine co-complexeswith the oligonucleotides of the invention may be prepared and used asdescribed to transfect hepatocytes in vivo.

The dsRNA oligonucleotides of the invention may be provided exogenouslyto a target hepatocyte, e.g., prepared outside the cell and deliveredinto a mammalian hepatocyte. Alternatively, a dsRNA may be producedwithin the target cell by transcription of a nucleic acid moleculecomprising a promoter sequence operably linked to a sequence encodingthe dsRNA. In this method, the nucleic acid molecule is contained withina non-replicating linear or circular DNA or RNA molecule, or iscontained within an autonomously replicating plasmid or viral vector, oris integrated into the host genome. Any vector that can transfect ahepatocyte may be used in the methods of the invention. Preferredvectors are viral vectors, including those derived fromreplication-defective hepatitis viruses (e.g., HBV and HCV),retroviruses (see, e.g., WO89/07136; Rosenberg et al., N. Eng. J. Med.323(9):570-578, 1990), adenovirus (see, e.g., Morsey et al., J. Cell.Biochem., Supp. 17E, 1993; Graham et al., in Murray, ed., Methods inMolecular Biology: Gene Transfer and Expression Protocols. Vol. 7,Clifton, N.J.: the Human Press 1991: 109-128), adeno-associated virus(Kotin et al., Proc. Natl. Acad. Sci. USA 87:2211-2215, 1990),replication defective herpes simplex viruses (HSV; Lu et al., Abstract,page 66, Abstracts of the Meeting on Gene Therapy, Sep. 22-26, 1992,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), and anymodified versions of these vectors. Methods for constructing expressionvectors are well known in the art (see, e.g., Molecular Cloning: ALaboratory Manual, Sambrook et al., eds., Cold Spring Harbor Laboratory,2nd Edition, Cold Spring Harbor, N.Y., 1989).

Appropriate regulatory sequences can be inserted into the vectors of theinvention using methods known to those skilled in the art, for example,by homologous recombination (Graham et al., J. Gen. Virol. 36:59-72,1977), or other appropriate methods (Molecular Cloning: A LaboratoryManual, Sambrook et al., eds., Cold Spring Harbor Laboratory, 2ndEdition, Cold Spring Harbor, N.Y., 1989).

Upon assembly of a recombinant DNA plasmid dsRNA expression vector onthe invention, bacteria are used as “factories” to produce largequantities of the final vector. The E. coli bacterium is frequently usedfor plasmid fermentation, and it may be advantageous to employ for thispurpose E. coli strains having a reduced genome as described in, e.g.,Blattner et al., Published U.S. Patent Application No. 2005/0032225, theteaching of which is incorporated herein by reference. The vectormanufactured in this manner, isolated and purified according to methodsknown in the art, can be introduced into living cells with a variety ofmethods, collectively known as “transfection”, including the methods andcompositions described above. Once inside the cell, the promoterelements are recognized by the cellular machinery available for genetranscription and the RNA effector molecules, e.g., shRNAs, will beproduced.

Other bacterial strains that may be advantageous for propagating aplasmid expression vector of the invention include the E. coli GT116Competent Cells available commercially from InvivoGen, San Diego, Calif.GT116 is a sbcCD deletion strain specifically engineered to support thegrowth of plasmid DNAs carrying hairpin structures, such as the plasmidsof the invention engineered to express one or more dsRNA effectormolecules which are hairpin RNAs. Hairpin structures are known to beunstable in E. coli due to their elimination by a protein complex calledSbcCD that recognizes and cleaves hairpins (Connelly et al., Proc. Natl.Acad. Sci. USA 95:7969-74 (1998)). The sbcCD and sbcD genes are deletedin E. coli GT116, which improves its utility for cloning plasmids withhairpin or other palindrome-containing structures.

Promoters

Promoters are inserted into the vectors so that they are operablylinked, typically but not invariably, 5′ to the nucleic acid sequenceencoding the dsRNA oligonucleotide. Any promoter that is capable ofdirecting initiation of transcription in a eukaryotic cell may be usedin the invention. For example, non-tissue-specific promoters, such asthe cytomegalovirus (DeBernardi et al., Proc. Natl. Acad. Sci. USA88:9257-9261, 1991, and references therein), mouse metallothionine Igene (Hammer, et al., J. Mol. Appl. Gen. 1:273-288, 1982), HSV thymidinekinase (McKnight, Cell 31:355-365 1982), and SV40 early (Benoist et al.,Nature 290:304-310, 1981) promoters may be used. Non-tissue-specificpromoters may be used in the invention, as expression of HBV and/or HCVdsRNA in non-liver cells directed by the non-tissue-specific promotersshould be harmless to the non-liver cells, because of the specificity ofthe HBV and HCV dsRNAs of the invention for viral sequences. However,preferred promoters for use in the invention are hepatocyte-specificpromoters, the use of which ensures that the RNAs are expressedprimarily in hepatocytes. Preferred hepatocyte-specific promotersinclude, but are not limited to the albumin, alpha-fetoprotein,alpha-1-antitrypsin, retinol-binding protein, and asialoglycoproteinreceptor promoters. Viral promoters and enhancers, such as those fromcytomegalovirus, herpes simplex viruses (types I and II), hepatitisviruses (A, B, and C), and Rous sarcoma virus (RSV; Fang et al.,Hepatology 10:781-787, 1989), may also be used in the invention.

dsRNA expression vectors may include promoters for RNA polymerase I, RNApolymerase II including but not limited to HCMV, SCMV, MCMV, RSV, EF2a,TK and other HSV promoters such as ICP6, ICP4 and ICP0 promoters, HBVpregenomic promoter, RNA pol III promoter, especially type 3 RNApolymerase III promoters, including but not limited to 7SK, U6, and H1,and tRNA promoters, as well as mitochondrial light and heavy strandpromoters. Desirably, the dsRNA expression vector comprises at least oneRNA polymerase II promoter, for example, a human CMV-immediate earlypromoter (HCMV-IE) or a simian CMV (SCMV) promoter, at least one RNApolymerase I promoter, or at least one RNA polymerase III promoter. Thepromoter may also be a T7 promoter, in which case, the cell furthercomprises T7 RNA polymerase. Alternatively, the promoter may be an SP6promoter, in which case, the cell further comprises SP6 RNA polymerase.The promoter may also be one convergent T7 promoter and one convergentSP6 RNA promoter. A cell may be made to contain T7 or SP6 polymerase bytransforming the cell with a T7 polymerase or an SP6 polymeraseexpression plasmid, respectively. In some embodiments, a T7 promoter ora RNA polymerase III promoter is operably linked to a nucleic acid thatencodes a short dsRNA (e.g., a dsRNA that is less than 200, 150, 100,75, 50, or 25 basepairs in length). In other embodiments, the promoteris a mitochondrial promoter that allows cytoplasmic transcription of thenucleic acid in the vector (see, for example, the mitochondria)promoters described in WO 00/63364, filed Apr. 19, 2000, and inWO/US2002/00543, filed 9 Jan. 2001). Alternatively, the promoter is aninducible promoter, such as a lac (Cronin et al. Genes & Development 15:1506-1517, 2001), ara (Khlebnikov et al., J Bacteriol. 2000 December;182(24):7029-34), ecdysone (Rheogene website), RU48 (mefepristone)(corticosteroid antagonist) (Wang X J, Liefer K M, Tsai S, O'Malley B W,Roop D R, Proc Natl Acad Sci USA. 1999 Jul. 20; 96(15):8483-8), or tetpromoter (Rendal et al., Hum Gene Ther. 2002; 13(2):335-42 andLarnartina et al., Hum Gene Ther. 2002; 13(2):199-210) or a promoterdisclosed in WO 00/63364, filed Apr. 19, 2000. Also useful in themethods and compositions of the invention are the structural andchimeric promoters taught in U.S. Ser. No. 60/464,434, filed 22 Apr.2003. See also the promoter systems taught in Pachuk, C., andSatishchandran, C., “Multiple-Compartment Eurkaryotic ExpressionSystems,” U.S. Provisional Application No. 60/497,304, filed 22 Aug.2003, which are considered particularly desirable in the methods andcompositions of the invention.

Liver specific promoters useful in dsRNA expression constructs of theinvention include the albumin promoter, the alpha-fetoprotein promoter(especially in liver cancer cells), the alpha-1-antitrypsin promoter,hepatitis B promoters, e.g., hepatitis B promoters including promotersfor the antigen genes, including core, e antigen, polymerase and Xprotein.

T7 Promoter/T7 Polymerase Expression Systems

A desirable method of the invention utilizes a T7 dsRNA expressionsystem to achieve cytoplasmic expression of dsRNA, (e.g., long or shortdsRNA molecules) in vertebrate cells (e.g., mammalian cells). The T7expression system utilizes the T7 promoter to express the desired dsRNA.Transcription is driven by the T7 RNA polymerase, which can be providedon a second plasmid or on the same plasmid. Bacteriophage T7 RNApolymerase (T7 Pol) is the product of T7 gene 1, which can recognize itsresponsive promoter sequence specifically and exhibit a hightranscriptase activity. The complete sequence of the T7 genome, withdetailed information about the different regions of the bacteriophage,including promoter sequences, is disclosed in Dunn & Studier, 1983, J.Mol. Biol. 166(4), 477-535 (see also NCBI ‘Genome’ database, AccessionNo. NC 00 1 604). The T7 promoter cannot be utilised by any other RNApolymerase than the polymerase of bacteriophage T7, which shows astringent specificity for the promoter (Chamberlin et al., 1970, Nature228:227-231). When utilizing the T7 expression system for expressingdsRNAs, for example, a first plasmid construct that expresses both asense and antisense strand under the control of converging T7 promotersand a second plasmid construct that expresses the T7 RNA polymeraseunder the control of an RSV promoter can be used. Both the dsRNA and theT7 RNA polymerase could advantageously be expressed from a singlebicistronic plasmid construct, particularly when the dsRNA is formedfrom a single RNA strand with inverted repeats or regions ofself-complementarity that enable the strand to assume a stem-loop orhairpin structure with an at least partially double stranded region.Individual sense and antisense strands which self assemble to form adsRNA can be synthesized by a single plasmid construct using, e.g.,converging promoters such as bacteriophage T7 promoters placedrespectively at the 5′ and 3′ ends of the complementary strands of aselected sequence to be transcribed. See also, e.g., the teaching of WO0063364, with respect to T7 dsRNA expression systems, as well as U.S.Ser. No. 60/399,998P, filed 31 Jul. 2002 and U.S. Ser. No. 60/419,532,filed 18 Oct. 2002.

Therapeutic Compositions of the Invention

The dsRNAs of the invention, and the recombinant vectors containingnucleic acid sequences encoding them, may be used in therapeuticcompositions for preventing or treating HCV and/or HBV infection. Thetherapeutic compositions of the invention may be used alone or inadmixture, or in chemical combination, with one or more materials,including other antiviral agents. Currently, lamivudine, adefovirdipivoxil, and interferon alpha have been approved for treatment of HBV,and it is anticipated that the compositions of the invention may be usedin combination with these and other drugs that are active against HBV,including emtricitabine (FTC) and entecavir. Since dsRNAs against HBVand/or HCV act through a novel mechanism (dsRNA-mediated genesilencing/RNAi), combination therapy of the agents of the invention andother antivirals is expected to significantly increase the efficacy oftherapy while substantially reducing the development of drug resistance,e.g., the development of lamivudine resistance, a problem of majorconcern with long term lamivudine therapy. Currently, interferon andribavirin are licensed for treatment of HCV, and as for HBV, it isanticipated that the compositions of the invention may be used incombination with these and other drugs that are active against HCV.Specific dosage regimens involving therapy with such multiple agents canbe determined through routine experimentation by those of ordinary skillin the art of clinical medicine.

Formulations will desirably include materials that increase thebiological stability of the oligonucleotides or the recombinant vectors,or materials that increase the ability of the therapeutic compositionsto penetrate hepatocytes selectively. The therapeutic compositions ofthe invention may be administered in pharmaceutically acceptablecarriers (e.g., physiological saline), which are selected on the basisof the mode and route of administration, and standard pharmaceuticalpractice. One having ordinary skill in the art can readily formulate apharmaceutical composition that comprises an oligonucleotide or a geneconstruct. In some cases, an isotonic formulation is used. Generally,additives for isotonicity can include sodium chloride, dextrose,mannitol, sorbitol and lactose. In some cases, isotonic solutions suchas phosphate buffered saline are preferred. Stabilizers include gelatinand albumin. In some embodiments, a vasoconstriction agent is added tothe formulation. The pharmaceutical preparations according to thepresent invention are provided sterile and pyrogen free. Suitablepharmaceutical carriers, as well as pharmaceutical necessities for usein pharmaceutical formulations, are described in Remington: The Scienceand Practice of Pharmacy (formerly Remington's Pharmaceutical Sciences),Mack Publishing Co., a standard reference text in this field, and in theUSP/NF.

Routes of administration include, but are not limited to, intramuscular,intraperitoneal, intradermal, subcutaneous, intravenous,intraarterially, intraoccularly and oral as well as transdermally or byinhalation or suppository. Preferred routes of administration includeintravenous, intramuscular, oral, intraperitoneal, intradermal,intraarterial and subcutaneous injection.

Targeted transfection of hepatocytes in vivo for delivery of dsRNAsagainst HBV and/or HCV may be accomplished through IV injection with acomposition comprising a DNA or RNA expression vector as describedherein complexed with a mixture (e.g., a 35%/65% ratio) of a lactosylspermine (mono or trilactosylated) and cholesteryl spermine (containingspermine to DNA at a charge ratio of 0.8). Such compositions areespecially useful for pharmaceutical applications and may readily beformulated in a suitable sterile, non-pyrogenic vehicle, e.g., bufferedsaline for injection, for parenteral administration, e.g., IV (includingIV infusion), IM, SC, and for intraperitoneal administration, as well asfor aerosolized formulations for pulmonary delivery via inhalation. Incertain formulations, a DNA expression construct of the invention may becomplexed with an endosomolytic spermine such cholesteryl sperminealone, without a targeting spermine; some routes of administration, suchas intraperitoneal injection or infusion, may achieve effective hepaticdelivery and transfection of a DNA construct of the invention, andexpression of RNA effector molecules, e.g., multiple dsRNA hairpinseffective against HBV and/or HCV.

Intraperitoneal administration (e.g., ultrasound guided intraperitonealinjection) of a sterile pharmaceutical composition comprising dsRNAeffector molecules and/or dsRNA expression constructs which providedsRNA effector molecules against HBV and/or HCV in a speciallyformulated delivery vehicle may be an advantageous route of delivery topromote uptake by liver cells, including hepatocytes. In somecompositions the dsRNA expression construct may be complexed with anappropriate transfection enhancing agent, e.g., with a mixture of alactosyl spermine (mono or trilactosylated) and cholesteryl spermine, orin other compositions with an endosomolytic spermine such cholesterylspermine alone, without a targeting spermine. The volume, concentration,and formulation of the pharmaceutical composition as well as the dosageregimen may be tailored specifically to maximize cellular delivery whileminimizing toxicity such as an inflammatory response. E.g, relativelylarge volumes (5, 10, 20, 50 ml or more) with corresponding lowconcentrations of active ingredients, as well as the inclusion of ananti-inflammatory compound such as a corticosteroid, may be utilized ifdesired. Formulations as known to those of skill in the art ofpharmaceutics may also be utilized to provide sustained release of dsRNAeffector molecules and/or dsRNA expression constructs of the invention.

dsRNAs or dsRNA expression constructs may be administered by meansincluding, but not limited to, traditional syringes, needlelessinjection devices, or “microprojectile bombardment gene guns”.Intraperitoneal injection may be accomplished, e.g., with a traditionalsyringe, with placement of the needle advantageously guided byultrasound or a similar technique. Alternatively, the dsRNA and/or dsRNAexpression construct may be introduced by various means into cells thatare removed from the individual. Such means include, for example, exvivo transfection, electroporation, microinjection and microprojectilebombardment. After the gene construct is taken up by the cells, they arereimplanted into the individual. It is contemplated that otherwisenon-immunogenic cells that have gene constructs incorporated therein canbe implanted into the individual even if the host cells were originallytaken from another individual.

In HBV infected individuals it is anticipated that the dsRNAcompositions of the invention may be useful as a pre-treatment inconjunction with therapeutic vaccination protocols designed to boostimmunity against the virus. It is also anticipated that the dsRNAcompositions of the invention may be useful for prophylaxis in a regimenof periodic administrations to individuals who because of occupationalor other potential for exposure are considered at high risk of exposureto HBV and/or HCV, e.g., fire, emergency, and health care personnel.Such an effective prophylactic regime may include administration of acomposition that provides an HBV and/or HCV dsRNA of the invention,e.g., weekly, biweekly, monthly, bimonthly, every three months, everyfour months, semi-yearly, or yearly, as can be determined throughroutine experimentation by those of skill in the art of clinicalmedicine. The ability of a dsRNA expression vector such as a plasmid orviral vector to express the dsRNAs of the invention over a relativelyprolonged period of time, expected to be in the range of weeks tomonths, is considered to be advantageous for this and otherapplications.

Dosage of dsRNAs

For administration of dsRNA (e.g., a short dsRNA to inhibit toxicity ora short or long dsRNA to silence a gene) to an animal, typically between10 mg to 100 mg, 1 mg to 10 mg, 500 μg to 1 mg, or 5 μg to 500 μg dsRNAis administered to a 90-150 pound person/animal (in order of increasingpreference). For administration of a vector encoding dsRNA (e.g., ashort dsRNA to inhibit toxicity or a short or long dsRNA to silence agene) to an animal, typically between 100 mg to 300 mg, 10 mg to 100 mg,1 mg to 10 mg, 500 μg to 1 mg, or 50 μg to 500 μg dsRNA expressionvector or construct is administered to a 90-150 pound person/animal (inorder of increasing preference). The dose may be adjusted based on theweight of the animal. In some embodiments, about 1 to 10 mg/kg or about2 to 2.5 mg/kg is administered. Other doses may also be used, asdetermined through routine experimentation by those of skill in the artof clinical medicine.

For administration in an intact animal, e.g., a human subject infectedwith HBV and/or HCV, between 1 mg and 100 mg, typically between 1 mg and10 mg, between 10 ng and 50 μg, between 50 ng and 100 ng, or between 100ng and 5 μg of dsRNA or DNA encoding one or more dsRNA effectormolecules is used. In desirable embodiments, approximately 10 μg of aDNA or 5 μg of dsRNA is administered to the animal. In a desirableembodiment, a pharmaceutical composition for parenteral administrationis prepared containing 10 mg of a plasmid dsRNA expression construct ofthe invention (in some formulations complexed with an appropriatetransfection facilitating agent such as cholesteryl spermine or amixture of cholesteryl spermine/trilactosyl spermine) in 25 ml of asuitable sterile vehicle for injection such as Normal Saline Injection,D5W, D5%/0.45% NaCl, D5%/0.2% NaCl, etc., and injected intraperitoneallyover 5 to 10 minutes, with needle placement guided by untrasound or asimilar technology. Administration may be repeated periodically, e.g.,weekly or monthly, as required. With respect to the methods of theinvention, it is not intended that the administration of dsRNA or DNAencoding dsRNA to cells or animals be limited to a particular mode ofadministration, dosage, or frequency of dosing; the present inventioncontemplates all modes of administration sufficient to provide a doseadequate to inhibit gene expression, prevent a disease, or treat adisease.

If desired, short dsRNA is delivered before, during, or after theexogenous delivery of dsRNA (e.g., a longer dsRNA) that might otherwisebe expected to induce cytotoxicity. See the teaching of U.S. Ser. No.10/425,006, filed 28 Apr. 2003, “Methods of Silencing Genes WithoutInducing Toxicity”, Pachuk.

Therapeutic Advantages of the Invention as it Relates to WorldwideDisease Incidence and Viral Variability

The mutability of the hepatitis C virus genome, and to a lesser butsignificant extent, the hepatitis B virus genome, has been describedabove as presenting challenges to the design of nucleic acid basedtherapeutics against these viral agents. The inventors havepainstakingly aligned thousands of individual HCV and HBV sequences,originally deriving from thousands of human viral isolates from widelydivergent geographic areas worldwide. In doing so, the instant inventionhas identified and specified preferred sequences which are utilizedsingly and in combination in dsRNA effector molecules which target theleast mutable regions of the genome of HCV and/or HBV.

The two-fold rationale for this has been discussed above, primarily interms of ensuring that during the course of infection of a patient withHBV or HCV, the therapeutic of the invention will remain potent againstthe virus even as it mutates during the course of disease in a givenpatient. However, the second part of this rationale for deriving andusing highly conserved sequences for design of dsRNA-¬based therapeuticapplications, is that this also increases the certainty that thetherapeutic dsRNA effector molecules of the invention, particularly themethods and compositions of the invention which utilize combinations ofhighly conserved sequences in dsRNA effector molecules against HBVand/or HCV, will be able to treat the viral infection present inindividuals from a global variety of different ancestries, geneticmakeup, and geographical distribution, which are known to manifest inclusters of viral variants based on such factors. Thus, a key feature ofthe therapeutic utility and novelty of this invention lies in the methodof derivation of the preferred sequences and embodiments, and not simplyin the demonstration that any particular HCV or HBV dsRNA sequence caninhibit viral replication of one or a few chosen viral isolates (ortheir cognate replicons) in a laboratory experiment, i.e., in a cellline or animal model, not necessarily reflective of the broad diversityof the HBV and/or HCV virus worldwide, or even in a particular infectedindividual over the course of infection.

For example, the hepatitis B virus has four subtypes of surface antigen,namely adw, ayw, adr and ayr. While lamivudine is considered aneffective therapy for chronic Hepatitis B, a recent study of HBVresistance demonstrated a 20-fold increase in resistance in the adwsubtype, compared to the ayw subtype. B Zollner et al. “20-fold Increasein Risk of Lamivudine [Epivir HBV] Resistance in Hepatitis B VirusSubtype adw”; The Lancet. 2001; 357: 934-935. In contrast to suchconventional antiviral agents, the dsRNA agents of the invention, (e.g.,dsRNA effector molecules and expression constructs of the invention,especially when used in combination as taught herein) which utilize HBVand/or HCV sequences highly conserved across such geographical geneticvariants are expected to exhibit highly advantageous antiviral activity.

Similarly, HCV is also known for having a wide range of geographicallydivergent viral genotypes, subtypes, quasispecies, with the followingcurrent general global patterns of genotypes and subtypes:

1a—mostly found in North & South America; also common in Australia

1 b—mostly found in Europe and Asia.

2a—is the most common genotype 2 in Japan and China.

2b—is the most common genotype 2 in the US and Northern Europe.

2c—the most common genotype 2 in Western and Southern Europe.

3a—highly prevalent here in Australia (40% of cases) and South Asia.

4a—highly prevalent in Egypt

4c—highly prevalent in Central Africa

5a—highly prevalent only in South Africa

6a—restricted to Hong Kong, Macau and Vietnam

7a and 7b—common in Thailand

8a, 8b & 9a—prevalent in Vietnam

10a & 11a—found in Indonesia

Accordingly, the highly conserved sequences of the invention, which areexpected to be conserved among most if not all of these divergent HCVgenotypes and subtypes worldwide, including 1a, 1b, 2a, 2b, and 2c, areconsidered highly effective therapeutics agents, e.g., when utilized asdsRNA effector molecules, especially combinations thereof, and dsRNAexpression vectors capable of expressing such dsRNAs.

Applicants specifically incorporate the entire content of all citedreferences in this disclosure. Further, when an amount, concentration,or other value or parameter is given as either a range, preferred range,or a list of upper preferable values and lower preferable values, thisis to be understood as specifically disclosing all ranges formed fromany pair of any upper range limit or preferred value and any lower rangelimit or preferred value, regardless of whether ranges are separatelydisclosed. Where a range of numerical values is recited herein, unlessotherwise stated, the range is intended to include the endpointsthereof, and all integers and fractions within the range. It is notintended that the scope of the invention be limited to the specificvalues recited when defining a range.

EXAMPLES

The following Examples are provided as illustrative only. All referencesmentioned within this disclosure are specifically incorporated herein byreference in their entirety.

Example 1 Silencing HBV Replication and Expression in a ReplicationCompetent Cell Culture Model

Brief description of cell culture model: A human liver-derived cell linesuch as the Huh7 cell line is transfected with an infectious molecularclone of HBV consisting of a terminally redundant viral genome that iscapable of transcribing all of the viral RNAs and producing infectiousvirus [1-3]. The replicon used in these studies is derived from thevirus sequence found in Gen Bank Accession V01460. Followinginternalization into hepatocytes and nuclear localization, transcriptionof the infectious HBV plasmid from several viral promoters has beenshown to initiate a cascade of events that mirror HBV replication. Theseevents include translation of transcribed viral mRNAs, packaging oftranscribed pregenomic RNA into core particles, reverse transcription ofpregenomic RNA, and assembly and secretion of virions and HBsAg(Hepatitis B Surface Antigen) particles into the media of transfectedcells. This transfection model reproduces most aspects of HBVreplication within infected liver cells and is therefore a good cellculture model with which to look at silencing of HBV expression andreplication.

Using this model, cells were co-transfected with the infectiousmolecular clone of HBV and various eiRNA constructs (dsRNA expressionconstructs). The cells were then monitored for loss of HBV expressionand replication as described below. Details on the vector and encodedRNAs used in this experiment are provided at the end of this example.

Experiment 1

The following is an example of an experiment that was performed usingeiRNA vectors (dsRNA expression vectors) encoding sequences derived fromGenBank accession number V01460. HBV sequences in these described eiRNAvectors were highly conserved sequences identified as describedelsewhere herein, which also exhibited activity as siRNAs (See, Pachuk,C., “Methods and Constructs for Evaluation of RNAi targets and EffectorMolecules,” PCT/US2004/005065, filed 25 Feb. 2004). The particular eiRNAbackbone vector used for this experiment was a proprietary vectorcontaining a U6 promoter to drive expression of the encoded RNAs. Eachvector. encoded only one short hairpin RNA (shRNA). The shRNA codingsequence was followed by an RNA pol III termination sequence. Sequencesof the U6 promoter, RNA pol In termination signal, and encoded shRNAsare all shown at the end of the example. Similar vectors containing U6promoters and RNA pol III termination signals are commercially availablesuch as the “siLentGene-2 Cloning Systems” vector from Promega, Inc.,Madison, Wis. One of ordinary skill in the art can also create themaccording to the information provided herein. It is expected thatsimilar results would also be obtained using other expression andpromoter systems especially those vectors with RNA pol III promotersthat are not U6, for example H1 promoters or 7SK promoters.

Experimental Procedure: Transfection

Huh7 cells cultured in RPMI-1640 media were seeded into six-well platesat a density of 3×10⁵ cells/well. All transfections were performed theday after cell seeding using Lipofectamine™ (InVitrogen, Carlsbad,Calif.) according to the manufacturer's directions. In this experiment,cells were transfected with 500 ng of the infectious HBV plasmid aywsubtype (“pHBV2”) (GenBank Accession #V01460) and 500 ng, 300 ng, 250ng, 120 ng, 100 ng, 50 ng, or 10 ng of an eiRNA construct. DNA was heldconstant/transfection at 2.5 μg by including an inert plasmid DNA,pGL3-Basic (Promega, Madison Wis.) in amounts that brought the total DNAin the transfection to 2.5 μg. For example, in transfections receiving500 ng of HBV DNA and 500 ng of an eiRNA construct, 1.5 μg pGL3 wasadded to the transfection. Prior to transfection, media was removed fromthe cells and the cells washed with Opti-MEM® (InVitrogen LifeTechnologies, Carlsbad, Calif.). 800 μl of Opti-MEM® was then added toeach well of cells followed by the addition of the transfection mix.Seventeen to nineteen hours post-transfection, the transfection mix andOpti-MEM® were removed from cells and replaced with 2 mL culturemedia/well. At 3, 6, and 10 days after transfection, the media wasremoved from cells and stored at −70° C. The media was replaced with 2mL of fresh culture media on days 3 and 6. All transfections werecarried out in duplicate. Two sets of control transfections were alsoperformed: HBV DNA alone (500 ng HBV DNA plus 2 μg pGL3) and HBV DNAwith a control eiRNA construct (500 ng HBV DNA, 1 μg control eiRNAconstruct, and 1.0 μg pGL3 DNA).

Monitoring Cells for Loss of HBV Expression.

Following transfection, cells were monitored for the loss or reductionin HBV expression and replication by measuring HBsAg secretion. Cellswere monitored by assaying the media of transfected cells (and a mediacontrol) at days 3, 6, and 10 post-transfection. The Auszyme® ELISA,commercially available from Abbott Labs (Abbott Park, III.), was used todetect surface Ag (sAg) according to the manufacturer's instructions.sAg was measured since surface Ag is associated not only with viralreplication but also with RNA polymerase II initiated transcription ofthe surface Ag cistron in the transfected infectious HBV clone and fromHBVcccDNA produced during infection in vivo. Since surface Ag synthesiscan continue with deleterious effects in the absence of HBV replication,it is important to down-regulate not only viral replication but alsoreplication-independent synthesis of sAg.

Results:

Cells transfected with the HBV-specific eiRNA constructs described atthe end of this example all induced a decrease in sAg levels relative tothe controls. The level inhibition is shown in the accompanying FIG. 2-8corresponding to data found in Tables 2-8. Note that the sequencesidentified as 788-808 and 807-827 only lowered surface Ag levels by 30%and 50% respectively at 500 ng doses. These are the only two eiRNAs thatdo not target the sAg mRNA; instead they target the 3.1 Kb HBV mRNAs andtherefore reduce sAg levels indirectly. The 30% to 50% reduction in sAgobserved when these other HBV RNAs are targeted is considered a strongindication that these eiRNA constructs are efficacious.

HBV-Specific eiRNAs Used in this Experiment

The eiRNA vectors encode the HBV sequences listed in Table 1. Thesequences are shown as well as their map coordinates on GenBankaccession number V01460. At the rightmost part of the table is the SEQID NO that these sequences map within. The sequence of the encoded RNAis 5′GGTCGAC (a sequence that is per se unimportant, but is derived fromthe polylinker sequence of the particular vector used) followed by afirst sense or antisense HBV sequence followed by the loop sequence(underlined in Table 1) followed by a second HBV sequence, which is thecomplement to the first HBV sequence. Note that the loop structure doesnot need to be a fixed sequence or length, and we have used several loopsequences with no significant impact on the functioning of the eiRNAconstruct. The second HBV sequence is followed by a string of Tresidues, e.g., 1, 2, 3, or more Ts, that function as the terminationsignal for RNA pol III.

TABLE 1 Maps HBV-AYW SEQ within coordi- ID SEQ ID nates* NO NO  788- 14CGTCTGCGAGGCGAGGGAGTTAGAGAACT  5,  708 TAACTCCCTCGCCTCGCAGACG  6,  7, or 8  807- 15 TTCTTCTTCTAGGGGACCTGCAGAGAACT  5,  827TGCAGGTCCCCTAGAAGAAGAA  6,  7, or  8 1291- 16AAGCCACCCAAGGCACAGCTTAGAGAACT  4 1311 TAAGCTGTGCCTTGGGTGGCTT 1299- 17CAAGGCACAGCTTGGAGGCTTAGAGAACT  4 1319 TAAGCCTCCAAGCTGTGCCTTG 1737- 18GGATTCAGCGCCGACGGGACGAGAGAACT 10 1757 TCGTCCCGTCGGCGCTGAATCC 1907- 19TTCCGCAGTATGGATCGGCAGAGAGAACT  3 1927 TCTGCCGATCCATACTGCGGAA 1912- 20CAGTATGGATCGGCAGAGGAGAGAGAACT  3 1932 TCTCCTCTGCCGATCCATACTG 1943- 21TCCACGCATGCGCTGATGGCCAGAGAACT  3 1963 TGGCCATCAGCGCATGCGTGGA 1991- 22TGCGTCAGCAAACACTTGGCAAGAGAACT  3 2011 TTGCCAAGTGTTTGCTGACGCA 2791- 23AAAACGCCGCAGACACATCCAAGAGAACT  2 2811 TTGGATGTGTCTGCGGCGTTTT 2791- 24AAAACACCACACACGCATCCAAGAGAACT  2 2811mut TTGGATGCGTGTGTGGTGTTTT 2912- 25TTGAGAGAAGTCCACCACGAGAGAGAACT  1 2932 TCTCGTGGTGGACTTCTCTCAA 2919- 26AAGTCCACCACGAGTCTAGACAGAGAACT  1 2939 TGTCTAGACTCGTGGTGGACTT  799- 49GCCTCGCAGACGAAGGTCTCAAGAGAACT  779 TTGAGACCTTCGTCTGCGAGGC *nucleotidecoordinates refer to Genbank accession number V01460.

A diagram of the transcribed RNA structure is shown in FIG. 9.

SEQ ID NO:13 is the nucleotide sequence of U6 promoter. Nucleotidesequence of RNA pol III terminator: 5′-TTTTT-3′.

The HBV sequitopes of Table 1 (without their respective complementsequence and without the “loop” or linker sequence utilized in a“hairpin” or stem-loop dsRNA effector molecule) are shown in Table 1Abelow. Each such HBV sequitope, together with its complementarysequence, optionally with an appropriate loop or linker sequence astaught herein, may be utilized in a dsRNA effector molecule of theinvention (e.g., a duplex dsRNA or hairpin dsRNA effector molecule, orencoded within a dsRNA expression vector). In one aspect, multiple(e.g., 2, 3, 4, 5, 6, or more) of such dsRNA short hairpin effectormolecules are encoded in a single expression vector, e.g., a plasmidexpression vector, each under the control of a different promoter, e.g.,a polymerase III promoter, as described elsewhere herein.

TABLE 1A HBV-AYW coordinates Genbank Maps accession SEQ within number IDConserved HBV SEQ ID V01460* NO Sequitope NO  788-808 50CGTCTGCGAGGCGAGGGAGTT  5,  6,  7, or  8  807-827 51TTCTTCTTCTAGGGGACCTGC  5,  6,  7, or  8 1291-1311 52AAGCCACCCAAGGCACAGCTT  4 1299 = 1319 53 CAAGGCACAGCTTGGAGGCTT  41737-1757 54 GGATTCAGCGCCGACGGGACG 10 1907-1927 55 TTCCGCAGTATGGATCGGCAG 3 1912-1932 56 CAGTATGGATCGGCAGAGGAG  3 1943-1963 57TCCACGCATGCGCTGATGGCC  3 1991-2011 58 TGCGTCAGCAAACACTTGGCA  3 2791-281159 AAAACGCCGCAGACACATCCA  2 2912-2932 60 TTGAGAGAAGTCCACCACGAG  12919-2939 61 AAGTCCACCACGAGTCTAGAC  1  799-779 62 GCCTCGCAGACGAAGGTCTCATables and Graphs.

HBsAg was measured as described above and plotted in FIG. 2-8corresponding to the data in Tables 2-8. The amount of eiRNA constructis shown in parentheses following the name of the eiRNA construct and isin μg amounts. For example, 2791(0.5) means that 0.5 μg or 500 ng ofeiRNA construct 2791-2811 (see Table 1) was used in the transfection.The percent inhibition relative to the control is also shown in thetables below and it is specific for the day 10 measurement. Note thatthe 4^(th) set of data in this example in which 1299 was evaluated at500 ng has only two timepoints, days 3 and 6, because the evaluation wasnot carried out at day 10. The percent inhibition for this experimentwas shown for day 6 data. Data is shown as raw OD data collected asdescribed by the manufacturer of the Auszyme ELISA assay kit used tomeasure sAg. Not shown are the 50 ng data for 2791-2811 and the 10 ngdata for 1907-1927. Each of these doses inhibited HBsAg expression byabout 50% relative to the control.

TABLE 2 % Inhibition relative to Day 3 Day 6 Day 10 control pHBV2 0.3391.88 3.268 — 2791(0.5) 0.101 0.263 0.333 89.8

TABLE 3 % Inhibition relative to Day 3 Day 6 Day 10 control pHBV2 1.1694.445 10.18 — 2791 (0.5) 0.442 0.743 1.3 87.2 2791Mut (0.5) 1.136 4.30510.595 —

TABLE 4 % Inhibition relative to Day 3 Day 6 Day 10 control pHBV2 0.3751.952 4.005 — 2791mut (1) 0.421 1.847 4.753 — HCV (1) 0.445 1.805 3.933—  788 (0.5) 0.255 1.195 2.778 30.6  807 (0.5) 0.254 1.326 2.015 49.71907 (0.25) 0.052 0.113 0.365 90.9 1912 (0.25) 0.138 0.208 0.517 87.11943 (0.25) 0.099 0.233 0.506 87.4 1991 (0.25) 0.075 0.152 0.291 92.72912 (0.25) 0.095 0.183 0.331 91.7

TABLE 5 % Inhibition relative to Day 3 Day 6 control pHBV2 0.474 1.513 —1299 (0.5) 0.439 0.699 53.8

TABLE 6 % Inhibition relative to Day 3 Day 6 Day 10 control pHBV2 0.331.617 2.88 — 2791 (0.3) 0.103 0.192 0.349 87.9 1737 (0.3) 0.051 0.0940.232 91.9 1291 (0.12) 0.239 0.587 1.195 58.5 1907 (0.12) 0.043 0.0860.356 87.6 2919 (0.12) 0.218 0.565 1.09 62.2

TABLE 7 % Inhibition relative to Day 3 Day 6 Day 10 control pHBV2 0.7412.53 5.383 — 2791 (0.3) 0.223 0.256 0.458 91.5 1737 (0.1) 0.212 0.3510.549 89.8 1907 (0.1) 0.067 0.149 0.468 91.3 1991 (0.1) 0.067 0.16 0.34593.6

TABLE 8 % Inhibition relative to Day 3 Day 6 Day 10 control pHBV2 0.8644.414 8.344 — 1907 (0.05) 0.17 0.538 1.396 83.3 2919 (0.1) 0.368 1.0441.908 77.1 1291 (0.2) 0.573 1.654 1.896 77.3

Experiment 2

Background: The same cell culture model was used to evaluate theadditive effects of adding two eiRNA constructs. In this experiment2791-2811 and 2919-2939 were evaluated. They were evaluated separatelyat two doses: 10 ng and 25 ng, and in combination at 10 ng (5 ng of2791-2811 plus 5 ng of 2919-2939) and at 25 ng (12.5 ng 2791-2811 plus12.5 ng 2919-2939). An additive effect is observed, for example, whenhalf the inhibition seen with 25 ng 2791-2811 plus half the inhibitionseen with 25 ng 2919-2939 is about equal to the inhibition seen of the25 ng combination dose. This is important because while one may not begaining inhibition over the use of a single eiRNA construct at the 25 ngdose, the use of two or more eiRNA sequences is very important inpreventing the generation of viral escape mutants.

Experimental Procedure: Transfection

Huh7 cells were seeded into six-well plates at a density of 3×10⁵cells/well. All transfections were performed the day after cell seedingusing Lipofectamine™ (InVitrogen) according to the manufacturer'sdirections. In this experiment, cells were transfected with 500 ng ofthe infectious HBV plasmid ayw subtype (GenBank Accession #V01460) and25 ng or 10 ng of two separate eiRNA constructs or a combination ofthese two eiRNA constructs at a total of 25 ng or 10 ng. DNA was heldconstant/transfection at 2.5 μg by including an inert plasmid DNA, pGL3,in amounts that brought the total DNA in the transfection to 2.5 μg. Forexample, in transfections receiving 500 ng of HBV DNA and 10 ng of aneiRNA construct, then 1.99 μg pLUC was added to the transfection. Priorto transfection, media was removed from the cells and the cells washedwith Opti-MEM® (InVitrogen Life Technologies). 800 μl of Opti-MEM® wasthen added to each well of cells followed by the addition of thetransfection mix. Seventeen to nineteen hours post-transfection, thetransfection mix and Opti-MEM® was removed from cells and replaced with2 mL culture media/well. At 4, 8, and 11 days after transfection, themedia was removed from cells and stored at −70° C. The media wasreplaced with 2 mL of fresh culture media on days 4 and 8. Alltransfections were carried out in duplicate. Two sets of controltransfections were also performed: HBV DNA alone (500 ng HBV DNA plus 2μg pGL3), and HBV DNA with a control eiRNA construct (500 ng HBV DNA,500 ng control eiRNA construct and 1.5 μg pGL3. DNA).

Results:

Results are shown in Table 9, and the corresponding graph found in FIG.10. Combining 2791-2811 and 2919-2939 showed at least equal effects toadministration of 2791-2811 or 2919-2939 alone. It is expected thatsimilar advantages will be seen by combining two or more dsRNAs directedto different HBV sequences from the same and/or different HBV genes.

TABLE 9 Day 4 Day 8 pHBV₂ 3.74 15.03 2791 @ 25 ng 2.49 9.63 2919 @ 25 ng2.55 10.07 2791 + 2919 @ 25 ng 2.73 10.91

Experiments 3 and 4

Silencing of HBV in a Mouse Model.

Summary: Two of the eiRNA vectors described in confirmatory experiment 1were assessed for their ability to silence an HBV replicon in a mousemodel. These vectors were the 2791-2811 and the 1907-1927 vectors. Bothvectors were found to silence HBV in the mouse model to a similar extentas they silenced in the cell culture model. The ability to silence thisHBV replicon in mice by other therapeutics has been demonstrated to be apredictor of human efficacy [4].

Animal Model Background:

Chimpanzees represent the only animal model in which to study human HBVinfectivity. A mouse model is available, however, in which HBVexpression and replication occur. This model has been invaluable for theevaluation of anti-HBV therapeutic agents not only targeted againstviral replication but also against RT-independent expression of antigen.In this model, replication competent HBV is expressed transiently fromepisomal HBV DNA. This model is created by introducing replicationcompetent HBV DNA into mouse liver by hydrodynamic delivery [1].

The aim of the following experiment was to test two of the vectorsencoding HBV-specific sequences evaluated in Experiment 1 for efficacyin a mouse model even though there were not expected to beHBV-sequence-related efficiency differences between the cell culture andmouse models. This experiment utilized hydrodynamic delivery as a methodto co-deliver replication competent HBVayw plasmid (Example 1,confirmatory experiment 1) with an effector HBV-specific eiRNAexpression vector. Hydrodynamic delivery is ideal for these firststudies because it results in efficient delivery of nucleic acid to theliver [5].

Experiments Hydrodynamic Delivery Studies: Experiment 3

All animals were hydrodynamically injected with 7.5 pg infectious HBVaywplasmid (described in confirmatory Example 1). Following internalizationinto hepatocytes and nuclear localization, transcription of HBVaywplasmid from several viral promoters has been shown to initiate acascade of events that mirror HBV replication [1]. These events includetranslation of transcribed viral mRNAs, packaging of transcribedpregenomic RNA into core particles, reverse transcription of pregenomicRNA, and assembly and secretion of virions and HBsAg particles into thesera of injected animals. Experimental animals were co-injected with 10μg 2791-2811. A second group of control animals were injected with 10 pgof an irrelevant eiRNA construct. All animals were also co-injected with2.5 μg of a GFP reporter plasmid (Clontech, Palo Alto, Calif.).Expression of GFP mRNA in the livers of injected mice served as acontrol to normalize results against the mouse model transfectionefficiency. Total DNA injected in animals was kept at a constant 20 μgby including pGL3, an inert filler DNA (Promega, Madison, Wis.). All DNAwas formulated and injected according to the methods described in Yanget al. [1]. There were 5 animals per group. The DNAs and amounts of DNAinjected per animal are shown in Table 10.

TABLE 10 GFP Group HBV DNA DNA eiRNA pGL3 1 7.5 μg 2.5 μg 10 μg 2791 0μg 2 7.5 μg 2.5 μg 10 μg control 0 μg 3 7.5 μg 2.5 μg  0 μg 10 μg 

Timepoints of analysis were selected based on published results from Dr.Chisari's laboratory [1], which detail the kinetics of HBVayw plasmidreplication in mice following hydrodynamic delivery. Serum was assayedfor the presence of HBsAg on days 1, 2, 3, and 4 post-injection. Assayswere performed as described for the cell culture model of HBVreplication. The presence of HBV RNA in liver samples was ascertained byNorthern blot analysis on day 2 following injection using proceduresdeveloped in Dr. Chisari's laboratory [1] and normalized to endogenousGAPDH RNA levels and GFP mRNA levels using conventional techniques, or aquantitative RT-PCR assay for HBV RNAs containing sAg coding sequencesusing standard techniques. RT-PCR is more quantitative than NorthernBlot analysis and has a larger dynamic window than does Northern Blotanalysis.

Downregulation of both HBV RNA by Northern Blot analysis and HBsAg wereseen in mice injected with 2791-2811. See FIG. 11. Also not shown,quantitative RT-PCR demonstrated the presence of 867 HBV RNA moleculesin the livers of control mice and 57 molecules of HBV RNA in 2791-2811treated mice, a 15-fold downregulation.

TABLE 11 HBV Group Std. Group Mouse 3.5 kb 2.1 kb GFP total HBV/GFPAverage Dev 10 μg 1 182360 1440614 4044344 1622974 4.0 3.0 0.76 2791 2392294 3161703 9954889 3553997 3.6 3 268673 3114347 15317275 3383020 2.24 394799 3909096 16806285 4303895 2.6 5 362182 4439430 18306755 48016122.6 HBV 21 2412562 8720964 3860082 11133526 28.8 17.4 7.40 only 222170741 7958388 6110744 10129129 16.6 23 2713213 12060855 963340414774068 15.3 24 1924373 7243024 11042915 9167397 8.3 25 1464641 57262173968243 7190858 18.1

TABLE 12 HBsAG (ng/ml) NUC5_HBsAg d1 d2 d3 d4 HBV  2 2810 6793 8422 8517 3 2344 8332 8089 8743  4 1684 8788 9064 8876  5 2318 9378 8597 8480 291066 5038 5153 5925 grp ave=> 2044 7666 7865 8108 Std Dev=> 678 17541556 1231 eiHCV  6 2554 8048 9233 8870  9 2267 8420 9535 8338 10 17048258 8761 7840 30 1362 4171 5406 4920 grp ave=> 1972 7224 8234 7492 StdDev=> 538 2041 1912 1765 2791 11 1262 2823 2276 2080 12 1222 2549 28581593 14 1056 1933 1143 792 15 1275 8320 1920 2068 27 779 4771 3782 1252grp ave=> 1119 4079 2396 1557 Std Dev=> 209 2598 993 551

Hydrodynamic Delivery Studies: Experiment 4

This experiment was similar to the Experiment 3 of Example 1 except thattwo eiRNA constructs were evaluated: 2791-2811 and 1907-1927. In thisexperiment, HBsAg was measured on days 1 and 4 using the assay alreadydescribed herein.

TABLE 13 HBsAG (ng/ml) NUC6_HBsAg d1 d4 HBV  2 6147  36,953   3 6234 42,542   4 4658 33,061   5 5077  29,389  grp ave=> 5529  35486 Std Dev=> 784   5627 eiHCV  6 1901 11,236  7 6286{circumflex over ( )}29,637{circumflex over ( )}  8 1023  6,345 grp ave=> 3070  15739 StdDev=> 2820  12282 2791 11 3966   5009 13 4705   7347{circumflex over( )} 14 2289   4538 15 2427   4217 grp ave=> 3347   5278 Std Dev=> 1182  1417 1907 16 4954   7203{circumflex over ( )} 18 2982  6917{circumflex over ( )} 19 3436   7568{circumflex over ( )} 20 2246  5135{circumflex over ( )} grp ave=> 3405   6706 Std Dev=> 1143   1081A Four-Promoter RNA Polymerase III-Based Expression Construct forProduction of shRNAs which Reduce Hepatitis B RNA Production andReplication.

As described in more detail in PCT/US05/29976 filed 23 Aug. 2005, aplasmid expression vector, pHB4, containing 4 polymerase III promotershort hairpin dsRNA expression cassettes was constructed. Eachexpression cassette included a polymerase III promoter operably linkedto a sequence encoding a short hairpin dsRNA, and a polymerase IIItermination sequence. The polymerase III promoters were U6, 7SK, and twocopies of a 7SK sequence variant promoter, designated 7SK-4A. Each shorthairpin dsRNA sequence comprised a double-stranded stem regionhomologous and complementary to a highly conserved HBV sequence astaught herein. The four dsRNA effector molecules comprised the sequencesof SEQ ID NO: 49; SEQ ID NO: 23; SEQ ID NO:19; and SEQ ID NO: 18; whichcomprise, respectively; the sequences of SEQ ID NO: 62; SEQ ID NO: 59;SEQ ID NO:55; and SEQ ID NO: 54. As described in more detail in Example1 of PCT/US05/29976, however, the sequence encoding the dsRNA hairpineffector molecule was inserted into an expression cassette of theplasmid expression vector at a restriction site which in effect resultedin several additional nucleotides being added to the 5′ end of theultimate transcript. The predicted transcript which includes the dsRNAhairpin actually contains additional 5′ and 3′ sequences: a 5′ leaderconsisting of 6 bases (e.g., the Sal I or Hind III or other chosenrecognition sequence), followed by the dsRNA hairpin sequences, followedby a short 3′ terminal U tract, usually two (1, 2, 3, or 4) U residuesincorporated during transcription termination. These differences inlength and composition of 5′ and 3′ transcript sequences flanking thedsRNA hairpin did not appear to adversely affect the ability of thedsRNA hairpin to effect dsRNA-mediated silencing, which suggests that,unlike synthetic dsRNA duplexes, endogenously expressed dsRNA hairpinconstructs are effective despite varying in a number of respects, e.g.,length of dsRNA “stem” between about 19-29 bp, length and composition ofsingle-stranded loop, presence or absence of additional short 5′ and/or3′ sequences.

A luciferase assay as taught in Example 1 of PCT/US05/29976 and in WO04/076629, published 10 Sep. 2004, “Methods and Constructs forEvaluation of RNAi Targets and Effector Molecules” indicated that all 4promoter/shRNA cassettes were active in silencing their target sequencesin a cell supplied with the vector and an assayable substrate. The IC50value decreased substantially when increasing from a one promoter/shRNAcassette vector to the pHB4 expression vector containing 4promoter/shRNA cassettes. The 1050 values may have also been affected bythe relative potency and transcription levels of each shRNA molecule,and did not reflect a simple relationship to the concentration of thevector only, which in effect behaved as four drugs after entering thecell and expressing the four encoded dsRNA molecules. In other words,the increased potency reflected not only the greater number of totalshRNA transcripts generated by the vector, but the also the individualpotency that each shRNA has to effect the reduction of sAg or eAgproduction via degradation of the target viral RNA molecules. Theprogressive addition of shRNA cassettes increased the potency of thevector in an apparently quantitative manner, and furthermore increasedthe pharmacological activity against the HBV target by inhibiting fourdistinct sites of the HBV target. It is important to recognize, however,that the ability of the multiple polymerase III expression constructs toexpress multiple individual antiviral dsRNA hairpin molecules is ofsignificant value in and of itself, not just because of associatedincreases in “potency”. Where the level of antiviral efficacy is high,the incremental quantitative increase in viral inhibition seen with eachadditional dsRNA molecule may be less important per se than the abilityof the constructs to deliver what is in effect a multi-drug regimen,with the inherent advantage of being highly resistant to the developmentof viral resistance.

The expression vectors, designed to deliver multiple dsRNA effectormolecules targeting highly conserved HBV and/or HCV polynucleotidesequences, when delivered to a virally infected cell, have the uniqueability to destroy the viral nucleic acid products directly. Moreover,inherent and integral to the design and intent of these multiplepromoter vectors (which express a plurality of different inhibitoryshort hairpin dsRNAs targeting different portions of the viral genome),is the property of generating multiple different viral antagonistssimultaneously. The antagonists (short hairpin dsRNA effector molecules)target different genome sequences in the viral genome. One of theseantagonists would probably be sufficient to disable the virus; however,the redundancy serves as a “backup” mechanism such that if the viralsequence mutates to render one antagonist inert, there are 2, 3, 4 ormore additional antagonists available. Additionally, by targetingmultiple sites in the viral genome, different DNA or RNA products of thevirus which play different roles in the disease pathology can beattacked at the same time.

In the case of Hepatitis B for example, in one embodiment, the instantinvention uses 4, 5, or more shRNA molecules selected from the followingsequences and other highly conserved HBV sequences as taught herein:e.g., “799” (SEQ ID No. 49); “1907” (SEQ ID No. 19); “2791” (SEQ ID No.23); “1737” (SEQ ID No. 18), “1991” (SEQ ID No. 22), “1943” (SEQ ID No.21). Other of the conserved HBV sequences disclosed herein, includingsequences of e.g., 19 to 29 nucleotides, which comprise all or part of“799”, “1907”, “2791”, “1737”, “1991”, or “1943”, may be selected forinclusion in dsRNA hairpin effector molecules to be expressed byexpression vectors comprising multiple promoters, including multiplepolymerase III expression vectors. Due to the nature of HBV geneexpression and overlapping transcriptional products this allowstargeting of multiple RNA transcripts as well as the replicativetemplate of the virus which will interfere with replication andexpression of more than one of the viral proteins. One of the shRNAmolecules, “1737” (SEQ ID No. 18) uniquely can disable the RNA encodinga product known as the X protein (HbX). Strong evidence exists in thebiomedical literature that the X protein plays a role in establishmentand/or maintenance of liver cancer. Because the existing drugs that canto some extent inhibit viral replication cannot eliminate the cell ofintegrated or other residual copies or portions of the viral genome,these drugs cannot shut off the production of HbX, even in patients“cured” of infectious HBV, and thus can not directly reduce anyincidence of cancer that is mediated by dormant HbX. Multiple anti-HBVdsRNA hairpin expression constructs of the present invention can attackboth the replication of the virus and the expression of all viralproteins, some which cause the inflammatory insult which results inhepatitis, and others such as HbX, which are believed to promotehepatocellular carcinoma via a distinct but not fully understoodmechanism. It is recognized that the principles taught herein can beused to design constructs of the invention specifically tailored totreat such “post infection” patients, which express dsRNAs against Hbxand any other residual HBV antigens.

While the HBV target sequences of the invention were chosen expressly torepresent those highly conserved or identical among a large number ofdifferent isolates (strains) of HBV, for reference purposes theidentified sequences, e.g., shRNA sequences, can be mapped back to HBVisolate AYW. It should be recognized, therefore, that the dsRNAs anddsRNA expression constructs of the invention are expected to beeffective not only against HBV AYW and related viral strains, butagainst nearly all if not all HBV strains encountered in infected humanpopulations in widely dispersed geographical areas.

Example 2 Hepatitis C— Sequences for RNAi Therapeutic DevelopmentExperiment 1

Brief Introduction

The hepatitis C virus (HCV) is the primary cause of non-A, non-Btransfusion-associated hepatitis and accounts for more than 200 millionhepatitis cases worldwide. The HCV genome has a high degree of sequencevariability. There are six major genotypes comprising more than fiftysubtypes and significant heterogeneity hallmarked by quasi-species hasbeen found within patients. Great progress in understanding HCVreplication has been made by using recombinant polymerases or cell-basedsubgenomic replicon systems. By using a replicon cell system,HCV-specific siRNA has been demonstrated to be able to suppress HCVprotein expression and RNA replication. Sequences of the 5′ NTR and bothstructural and nonstructural genes have been targeted successfully. Thehighly conserved nature of the 3′ NTR sequence makes it a highlyattractive target for siRNA based therapy. However, no study has beendone to examine the feasibility of using the 3′ NTR. Here we report thedesign and testing of several siRNAs that can inhibit HCV proteinexpression in the subgenomic replicon system. Exogenously synthesizedHCV-specific siRNAs were transfected into the HCV replicon cell line asdescribed below.

Cell Culture and Media:

The HCV replicon in hepatoma Huh7 cells was cultured in Dulbecco'sModified Eagle Media (“DMEM”) (Invitrogen) containing 10% fetal calfserum (Invitrogen), 1% penicillin-streptomycin, 1% non-essential aminoacids and 0.5 mg/mL Geneticin. Cells were grown to 75% confluency priorto splitting.

Western Blot Analysis:

Total cell lysates from replicon cells were harvested from repliconcells in 1×LDS Buffer (Invitrogen). The lysates were heated at 90° C.for 5 min in the presence of beta-mercaptoethanol before electrophoresison a 10% Tris-Glycine polyacrylamide gel (Invitrogen). The protein wastransferred to PVDF (Invitrogen) membrane. Following the transfer, themembrane was rinsed once with PBS containing 0.5% Tween-20 (PBS Tween)and blocked in PBS-Tween containing 5% non-fat milk for 1 hr. Afterwashing with PBS-Tween, the membrane was incubated with the primaryα-NS5A antibody (a gift from Dr. Chen Liu) at 1:1500 dilution for 1 hrat room temperature. Prior to incubation with HRP conjugated a-mouse IgGsecondary antibody (Amersham) diluted 1:5000, the blot was washed inPBS-Tween 20. Following the secondary antibody incubation, the blot waswashed again and treated with ECL (Amersham) according to themanufacturer's protocol.

Northern Blot:

Total cellular RNA was extracted by using the Rneasy® kit (Qiagen).Northern blot analysis was done according to the protocol of Guo et al.Briefly, 5 μg total RNA was electrophoresed through a 1.0% agarose gelcontaining 2.2 M formaldehyde, transferred to a nylon membrane andimmobilized by UV cross-linking (Stratagene). Hybridization was carriedout using α[³²P]CTP-labeled neomycin RNA in a solution containing 50%deionized formamide, 5×SSC (750 mM sodium chloride, 750 mM sodiumcitrate), Denhardt's solution, 0.02 M sodium phosphate (pH 6.8), 0.2%sodium dodecyl sulfate (“SDS”), 100 μg of sheared denatured salmon spermDNA/ml, and 100 pg of yeast RNA/ml, for 16 hr at 58° C. The membraneswere washed once in 2×SSC/0.1% SDS for 30 min at room temperature andtwice in 0.1×SSC/0.1% SDS for 30 min at 68° C. Membranes were exposed toX-ray film.

Transfection of siRNA into Replicon Cells:

For transfection of siRNA into replicon cells the Lipofectamine® 2000reagent (Invitrogen) was used according to the user manual. Briefly,2×10⁴ cells in 0.5 mL of DMEM was seeded in 24 well plates one daybefore the transfection. The indicated amount of siRNA was diluted in 50μL OptiMEM and mixed with diluted Lipofectamine® 2000 reagent (1 μL in50 μL of Optimem). The mixture was incubated at room temperature for 20min before being applied onto the cell monolayer. 48-72 hr aftertransfection, cells were washed in PBS and lysed in 100 μL. SDS samplebuffer.

TABLE 14 SEQ siRNA ID number NO HCV sequence  #12 28GCTAAACACTCCAGGCCAATACCTGTCTC  #22 29 TCCTTTGGTGGCTCCATCTTACCTGTCTC  #3230 GCTCCATCTTAGCCCTAGTCACCTGTCTC  #42 31 TCTTAGCCCTAGTCACGGCTACCTGTCTC #52 32 CCTAGTCACGGCTAGCTGTGACCTGTCTC  #62 33CTAGTCACGGCTAGCTGTGAACCTGTCTC  #72 34 CGTGAGCCGCTTGACTGCAGACCTGTCTC  #8235 GCTGATACTGGCCTCTCTGCACCTGTCTC #102 36 ACTGGCCTCTCTGCAGATCAACCTGTCTC

Several short duplex dsRNAs comprising the HCV sequences identifiedabove in Table 14 (in each case, the first 21 bases constitute conservedHCV sequences of the invention, followed by an 8-base “adapter”sequence, “CCTGTCTC”, appended from the Ambion kit used in synthesis,but which do not appear in the dsRNA effector molecules) targeting the3′UTR; siRNA #12 targeting the HCV NS5B gene (positive control); theidentified HCV core siRNA (positive control); and the identified laminsiRNA (negative control) were synthesized using the Silencer siRNAconstruction kit, Catalog #1620 (Ambion Inc., Austin, Tex.). DNAoligonucleotides were synthesized by IDT (Coralville, Iowa).

TABLE 14A SEQ siRNA ID number NO HCV sequence  #12 63GCTAAACACTCCAGGCCAATA  #22 64 TCCTTTGGTGGCTCCATCTTA  #32 65GCTCCATCTTAGCCCTAGTCA  #42 66 TCTTAGCCCTAGTCACGGCTA  #52 67CCTAGTCACGGCTAGCTGTGA  #62 58 CTAGTCACGGCTAGCTGTGAA  #72 69CGTGAGCCGCTTGACTGCAGA  #82 70 GCTGATACTGGCCTCTCTGCA #102 71ACTGGCCTCTCTGCAGATCAA

Several siRNAs comprising the HCV sequences identified above in Table 14targeting the 3′UTR; siRNA #12 targeting the HCV NS5B gene (positivecontrol); the identified HCV core siRNA (positive control); and theidentified lamin siRNA (negative control) were synthesized using theSilencer siRNA construction kit, Catalog #1620 (Ambion Inc., Austin,Tex.). DNA oligonucleotides were synthesized by IDT (Coralville, Iowa).

Control siRNAs:

1. HCV core (positive control): SEQ ID NO:45

2. #12, shown in Table 14, targeting the HCV NS5B gene, also a positivecontrol

3. lamin sequence (negative control): SEQ ID NO:46

Three siRNAs were used as controls: siRNA targeting the cellular geneLamin for negative control; siRNA targeting the core sequence of HCV asa positive control; siRNA targeting the HCV NS5B gene as a positivecontrol. Two concentrations of each siRNA (9 and 20 pmole) were used andthe results were compared with transfection of no siRNA. Accordingly,the Western Blots in FIG. 13 represent 0, 9, and 20 pmoles of theidentified siRNAs. siRNA #22, 32, 42, 62, and 72 were notably active inrepressing HCV NS5A protein expression. Presumably, HCV RNA level isalso decreased based on the results obtained previously with positivecontrol siRNA for core. Several siRNAs had minimum effect at theconcentrations tested and should be evaluated at higher concentrations.These include #12 (targeting NS5B), #102, #52, and #82.

Experiment 2

Experiment 2 was performed as described in Experiment 1 of HepatitisC-Sequences for RNAi Therapeutic Development except that siRNAs R1-R8,comprising the sequences (and their complements) set forth in Table 15below, were used in transfections. The Western Blot assay performed herewas as described in Example 2, Experiment 1. The control HCV core siRNAused as a positive control is the siRNA described in the previous HCVExperiment 1. All siRNAs were transfected at concentrations of 0, 9, and20 pmole except the control “core” siRNA, which was transfected atlevels of 0, 3, and 9 pmole. R1, R2, R3, R5, R7, and R8 all exhibitedsignificant inhibition of HCV as can be seen in the Western Blot, FIG.14.

TABLE 15 SEQ ID siRNA NO HCV sequence R1 37 CTGGCCTCTCTGCAGATCAAG R2 38TGCAGAGAGTGCTGATACTGG R3 39 TGAGCCGCTTGACTGCAGAGA R4 40GAAAGGTCCGTGAGCCGCTT R5 41 TAGCTGTGAAAGGTCCGTGAG R6 42TTAGCCCTAGTCACGGCTAGC R7 43 TCCATCTTAGCCCTAGTCACG R8 44TTGGTGGCTCCATCTTAGCCC

All siRNAs evaluated map to the 3′UTR of the HCV genome and areconserved amongst HCV genotypes and quasi-species. SEQ ID NO:27represents this 101 nt sequence of the HCV 3′UTR, sometimes referred toas the “X” region.

Example 3 Silencing HBV Replication and Expression in a ReplicationCompetent Cell Culture Model

Brief Description of Cell Culture Model:

A human liver derived cell line such as the Huh7 cell line istransfected with an infectious molecular clone of HBV consisting of aterminally redundant viral genome that is capable of transcribing all ofthe viral RNAs and producing infectious virus [1-3]. The replicon usedin these studies is derived from the virus sequence found in Gen BankAccession #s V01460 and J02203. Following internalization intohepatocytes and nuclear localization, transcription of the infectiousHBV plasmid from several viral promoters has been shown to initiate acascade of events that mirrors HBV replication. These events includetranslation of transcribed viral mRNAs, packaging of transcribedpregenomic RNA into core particles, reverse transcription of pregenomicRNA, and assembly and secretion of virions and HBsAg (Hepatitis BSurface Antigen) particles into the media of transfected cells. Thistransfection model reproduces most aspects of HBV replication withininfected liver cells and is therefore a good cell culture model withwhich to look at silencing of HBV expression and replication.

In this model, cells are co-transfected with the infectious molecularclone of HBV and the individual effector RNA constructs to be evaluated.The cells are then monitored for loss of HBV expression and replicationas described below.

The following is an example of an experiment using eiRNA vectorsencoding sequences derived from SEQ ID NO:1 and SEQ ID NO:5. Theparticular eiRNA vectors for this experiment are T7 RNA polymerase-based(See, e.g., the teaching of WO 0063364, with respect to T7 dsRNAexpression systems, as well as U.S. Ser. No. 60/399,998P, filed 31 Jul.2002 and U.S. Ser. No. 60/419,532, filed 18 Oct. 2002) and encodehairpin RNA structures (especially desirable are, e.g., “forced” hairpinconstructs, partial hairpins capable of being extended by RNA-dependentRNA polymerase to form dsRNA hairpins, as taught in U.S. Ser. No.60/399,998P, filed 31 Jul. 2002 and PCT/US2003/024028, filed 31 Jul.2003, as well as the “udderly” structured hairpins (e.g., multi-hairpinlong dsRNA vectors and multi-short hairpin structures), hairpins withmismatched regions, and multiepitope constructs as taught in U.S. Ser.No. 60/419,532, filed 18 Oct. 2002, and PCT/US2003/033466, filed 20 Oct.2003). It is expected that similar results will be obtained using otherexpression and promoter systems, e.g., as described above, and/orvectors encoding alternative dsRNA structures (i.e. duplex).

Experimental Procedure: Transfection

Huh7 cells are seeded into six-well plates such that they are between80-90% confluency at the time of transfection. All transfections areperformed using Lipofectamine™ (Invitrogen) according to themanufacturer's directions. In this experiment, cells are transfectedwith 50 ng of the infectious HBV plasmid, 1 μg of a T7 RNA polymeraseexpression plasmid (description of plasmid below) 600 ng of an eiRNAvector encoding a hairpin RNA comprised of sequences derived from SEQ IDNO:1 (described below) and 600 ng of an eiRNA vector encoding a hairpinRNA comprised of sequences derived from SEQ ID NO:5 (described below).Control cells are transfected with 50 ng of the HBV plasmid and 1 μg ofthe T7 RNA polymerase expression plasmid. An inert filler DNA,pGL3-basic (Promega, Madison Wis.), is added to all transfections tobring total DNA/transfection up to 2.5 μg DNA.

Monitoring Cells for Loss of HBV Expression.

Following transfection, cells are monitored for the loss or reduction inHBV expression and replication by measuring HBsAg secretion andDNA-containing viral particle secretion. Cells are monitored by assayingthe media of transfected cells beginning at 2 days post dsRNAadministration and every other day thereafter for a period of threeweeks. The Auszyme ELISA, commercially available from Abbott Labs(Abbott Park, Ill.), is used to detect hepatitis B surface antigen(HBsAg). HBsAg is measured since HBsAg is associated not only with viralreplication but also with RNA polymerase II initiated transcription ofthe surface antigen cistron in the transfected infectious HBV clone.Since HBsAg synthesis can continue in the absence of HBV replication itis important to down-regulate not only viral replication but alsoreplication-independent synthesis of HBsAg. Secretion of virionparticles containing encapsidated HBV genomic DNA is also measured. Lossof virion particles containing encapsidated DNA is indicative of a lossof HBV replication. Analysis of virion secretion involves a techniquethat discriminates between naked, immature core particles and envelopedinfectious HBV virions [6]. Briefly, pelleted viral particles from themedia of cultured cells are subjected to Proteinase K digestion todegrade the core proteins. Following inactivation of Proteinase K, thesample is incubated with RQ1 DNase (Promega, Madison, Wis.) to degradethe DNA liberated from core particles. The sample is digested again withProteinase K in the presence of SDS to inactivate the DNase as well asto disrupt and degrade the infectious enveloped virion particle. DNA isthen purified by phenol/chloroform extraction and ethanol precipitated.HBV specific DNA is detected by gel electrophoresis followed by SouthernBlot analysis.

Results will desirably indicate a 70-95% decrease in both HBsAg andviral particle secretion in the media of cells transfected with the HBVplasmid, T7 RNA polymerase expression plasmid and eiRNA constructsrelative to cells transfected with only the HBV plasmid and T7 RNApolymerase expression plasmid.

Vectors Used in Experiment

Sequence of the T7 RNA Polymerase Gene

SEQ ID NO:47 represents the T7 RNA polymerase gene which is cloned intoa mammalian expression vector such as pCEP4 (Invitrogen, Carlsbad,Calif.). Cloning can be easily done by one skilled in the art. Oneskilled in the art would also be aware that a leader sequence with aKozak sequence needs to be cloned in directly upstream from the T7 RNApolymerase gene.

eiRNA Vector Encoding RNA Hairpin Derived from SEQ ID NO:1

The vector is T7-based as described above. The insert encodes aunimolecular hairpin comprised of sequences mapping from coordinate3004-2950 (about 55 bp) of GenBank accession #s V01460 and J02203. Oneregion of the hairpin encodes the sense version of the sequences and thesecond region of the hairpin encodes the antisense version of thissequence. Hairpins can easily be designed and made by those skilled inthe art.

eiRNA Vector Encoding RNA Hairpin Derived from SEQ ID NO:5

The vector is T7-based as described above. The insert encodes aunimolecular hairpin comprised of sequences mapping from coordinate730-786 of GenBank accession #s V01460 and J02203. The hairpin isdesigned as described for hairpin encoding sequences from SEQ ID NO:1.

Experiment 1

Rationale for Mouse Models:

Chimpanzees represent the only animal model in which to study human HBVinfectivity. Mouse models are available, however, in which human HBVexpression and replication occur. These models have been invaluable forthe evaluation of anti-HBV therapeutic agents and have been shown to bea predictor for the efficacy of these agents in humans [4]. The first ofthese models are transgenic mouse models, in which the HBV genome orselected HBV genes are expressed [7,8]. Because HBV is integrated intothe mouse genome, these animals serve as a model not only for viralreplication but also for RT-independent expression of antigen. A similarmodel exists in which replication competent HBV is expressed transientlyfrom episomal HBV DNA. This model is created by introducing replicationcompetent HBV DNA into mouse liver by hydrodynamic delivery [1]. Unlikethe transgenic animals, these mice are not immunotolerant to HBVantigens and immune-mediated clearance of HBV transfected hepatocytescan be studied.

Although woodchuck and duck models exist for the study of woodchuckhepatitis (WHBV) and duck hepatitis (DHBV) respectively, we have optednot to use these models for several reasons. 1) Human HBV cannot bestudied in these models. As we are ultimately interested indown-regulating expression of human HBV, use of these models would atsome point necessitate the re-design and evaluation of vectors and/orRNAs specific for human HBV. 2) the mice are isogenic and thereforenoise due to genetic variables within the system does not arise. 3)Unlike human HBV, there are no validated WHBV/DHBV cell culture modelsthat can be studied in parallel with their respective animal models.

The experiment described below utilizes hydrodynamic delivery as amethod to co-deliver replication competent HBVayw plasmid with thevarious effector dsRNA (eiRNA) expression vectors. Hydrodynamic deliveryis ideal for this experiment because it results in efficient delivery ofnucleic acid to the liver [5]. Combination of the dsRNA effector plasmidand replication competent HBV plasmid into the same formulationincreases the likelihood that both plasmids are taken up by the samecells. Because expressed effector dsRNA are present in the majority ofcells bearing the replicating HBV plasmid, observed results can beattributed to the performance of the effector plasmid rather than todifferences in delivery efficiencies. This experiment demonstrates onlythat a particular eiRNA is efficacious in an infected liver. Formulationand delivery are not addressed by this example. Formulation, dosing anddelivery of the eiRNA vector are enabled in the example in whichtransgenic mice are used.

Experimental Procedure

Control B10.D2 mice are hydrodynamically injected with an infectiousmolecular clone of HBV (ayw subtype) consisting of a terminallyredundant viral genome that is capable of transcribing all of the viralRNAs and producing infectious virus [1,2,3]. Following internalizationinto hepatocytes and nuclear localization, transcription of HBVaywplasmid from several viral promoters has been shown to initiate acascade of events that mirror HBV replication [1]. These events includetranslation of transcribed viral mRNAs, packaging of transcribedpregenomic RNA into core particles, reverse transcription of pregenomicRNA, and assembly and secretion of virions and HBsAg particles into thesera of injected animals. Animals are injected with four doses of theHBV replicon plasmid (1 μg, 3 μg, 5 and 10 μg). These doses are chosenbecause they represent non-saturating doses capable of elicitingdetectable expression of a reporter plasmid following hydrodynamicdelivery. Animals are co-injected with the effector dsRNA expressionvector (eiRNA) such that animals in each group receive a 10-19 μg doseof a particular effector construct(s) such that the total DNA dose is 20μg. For example in mice receiving the 3 μg dose of the HBV replicon, 17μg of the chosen eiRNA vector(s) is injected for a total of 20 μginjected DNA. The amount of this dose is therefore dependent upon thedose of HBV plasmid used. Control animals are injected with the HBVreplicon but not with an eiRNA vector. Control mice are insteadco-injected with an inert filler DNA, pGL3-basic (Promega, Madison,Wis.) such that the total amount of DNA in the formulation is 20 μg.eiRNA vectors in this study are the U6-based expression plasmids, e.g.,Ambion, Inc., Austin, Tex., USA. These vectors encode short hairpin RNAsderived from SEQ ID NO:1 and SEQ ID NO:4. The exact sequences encoded bythese vectors are described below. The vectors are co-injected in equalamounts (by weight). It is expected that similar results will beobtained using other expression and promoter systems as describedelsewhere herein and/or vectors encoding alternative structures (i.e.duplex).

Description of U6-based eiRNA vector encoding sequences derived from SEQID NO:1: vector encodes a hairpin containing sequences mapping tocoordinates 2905-2929 of accession #s V01460 and J02203 (i.e. thehairpin contains the sense and antisense version of this sequence,separated by a loop structure of TTCAAAAGA). Description of U6-basedvector sequences can be found in Lee et al. [9]. The second eiRNA vectorused in this experiment encodes a hairpin derived from SEQ ID NO:4 andencodes sequences mapping to coordinates 1215-1239 of Accession #V01460and J02203.

Liver samples are taken from injected animals on day 1 followinginjection and analyzed for the presence of HBV RNA. This time point hasbeen selected based on published results from Dr. Chisari's laboratorywhich detail the kinetics of HBVayw plasmid replication in micefollowing hydrodynamic delivery and demonstrates that peak RNAexpression occurs in the liver on day 1 following hydrodynamic delivery[1]. The presence of HBV RNA in liver samples is ascertained by Northernblot analysis. Liver tissue will be evaluated for the down-regulation ofHBV RNA expression. In addition, serum will be collected from day 4 micefor measurement of HBVsAg and DNA-containing viral particles. Assayswill be as described for the cell culture replicon experiment (Example3) and as in Yang et al. [1]. Each vector and control group will becomprised of 2 sets of animals, each set corresponding to a collectiontime point. There are 5 animals is each set.

Results:

Mice that are injected with the HBV replicon and the eiRNA constructswill have decreased HBV-specific RNA, and HBsAg and HBV viral particlesas compared to the control animals. In individual animals, decreaseswill range from about 70% to near 100%.

Experiment 2

Transgenic Mouse Studies: Background.

We will be using the HBV transgenic mouse model developed in Dr.Chisari's laboratory [8]. These mice replicate appreciable amounts ofHBV DNA and have demonstrated their utility as an antiviral screen thatis a predictor of human efficacy [4]. These animals are also ideal inthat they are a model for HBV-integrant-mediated expression of antigenand thus can serve as a model not only for viral replication but alsofor RT-independent expression of antigen. This is important as we areinterested in targeting not only viral replication butintegrant-mediated antigen expression as well.

These experiments differ from the hydrodynamic delivery experiments inthat the effector plasmids are administered to animals using clinicallyrelevant nucleic acid delivery methods. Effectiveness in this modeldemonstrates efficient delivery of the effector plasmids to mousehepatocytes.

Experiment

Mice described in reference [8] will be injected IV with a formulationcontaining the eiRNA vectors described in the hydrodynamic deliveryexample. These are the U6-based eiRNA vectors encoding hairpinscontaining sequences derived from SEQ ID NO:1 and SEQ ID NO:4.

Formulation of DNA to be Injected.

DNA is formulated with trilactosyl spermine and cholesteryl spermine asdescribed in PCT/US03/14288, “Methods for Delivery of Nucleic Acids”,Satishchandran, filed 6 May 2003. Briefly, three formulations are made,all using a charge ratio of 1.2 (positive to negative charge). However,it should noted that formulations with charge ratios between 0.8 and 1.2are all expected to exhibit efficacy. The DNA starting stock solutionfor each plasmid is 4 mg/ml. The two plasmid stock solutions are mixedtogether in equal amounts such that each plasmid is at 2 mg/ml. Thisplasmid mixture is used for the final formulating. Formulation is asdescribed in PCT/US03/14288 (above): Formulation A) 35% trilactosylspermine, 65% cholesteryl spermine, Formulation B) 50% trilactosylspermine, 50% cholesteryl spermine and Formulation C) 80% trilactosyslspermine, 20% cholesteryl spermine. All resultant formulations nowcontain each plasmid at 1 mg/ml.

Mice are IV injected with 100 μl formulated DNA. One group of micereceives Formulation A, a second group receives Formulation B and athird group receives Formulation C. Three groups of control mice aresimilarly injected with formulations containing a control DNA, pGL3Basic(Promega, Madison Wis.), Formulations D, E and F. Injections are carriedout once a day for four consecutive days. Injecting for only 1-3 days isefficacious, however, more robust efficacy is seen with a four dayinjection protocol.

Following administration, HBV RNA and serum levels of HBsAg and DNAcontaining viral particles will be quantitated on days 5 and 9 postfirst injection. All analyses will be as described for the hydrodynamicdelivery studies.

Results:

HBV-specific RNA levels, HBsAg and virus containing DNA particles willhave decreased relative to controls in the Formulation A, B and Cgroups.

Example #4 Silencing HBV Replication and Expression in a ReplicationCompetent Cell Culture Model

Brief Description of Cell Culture Model:

A human liver derived cell line such as the Huh7 cell line istransfected with an infectious molecular clone of HBV consisting of aterminally redundant viral genome that is capable of transcribing all ofthe viral RNAs and producing infectious virus [1-3]. The replicon usedin these studies is derived from the virus sequence found in Gen BankAccession AF090840. Following internalization into hepatocytes andnuclear localization, transcription of the infectious HBV plasmid fromseveral viral promoters has been shown to initiate a cascade of eventsthat mirror HBV replication. These events include translation oftranscribed viral mRNAs, packaging of transcribed pregenomic RNA intocore particles, reverse transcription of pregenomic RNA, and assemblyand secretion of virions and HBsAg particles into the media oftransfected cells. This transfection model, reproduces most aspects ofHBV replication within infected liver cells and is therefore a good cellculture model with which to look at silencing of HBV expression andreplication.

In this model, cells were co-transfected with the infectious molecularclone of HBV and an eiRNA construct. The cells were then monitored forloss of HBV expression and replication as described below.

The following is an example of an experiment that was performed using aneiRNA vector encoding sequences derived from both SEQ ID NO:1 and SEQ IDNO:2. The particular eiRNA vector used for this experiment is T7 RNApolymerase-based and encodes a duplex RNA of about 650 by (See e.g., WO00/63364, filed Apr. 19, 2000). It is expected that similar resultswould be obtained using other expression and promoter systems asdescribed elsewhere herein and/or vectors encoding alternativestructures (i.e. duplex).

Experimental Procedure: Transfection

Huh7 cells were seeded into six-well plates such that they were between80-90% confluency at the time of transfection. All transfections wereperformed using Lipofectamine™ (InVitrogen) according to themanufacturer's directions. In this experiment, cells were transfectedwith A) 50 ng of the infectious HBV plasmid adw subtype, 1 μg of a T7RNA polymerase expression plasmid (description of plasmid in Example 3),and 1.5 μg of the HBV-specific eiRNA vector (described below); B) 50 ngof the infectious HBV plasmid, 1 μg of the T7 RNA polymerase expressionplasmid and 1.5 μg of an irrelevant dsRNA expression vector; C) 125 ngof the infectious HBV plasmid, 1 μg of the T7 RNA polymerase expressionplasmid and 1.4 μg of the HBV-specific eiRNA vector; and D) 125 ng ofthe infectious HBV plasmid, 1 μg of the T7 RNA polymerase expressionplasmid and 1.4 μg of an irrelevant dsRNA expression vector. Alltransfections were carried out in duplicate. In this experimenttransfections B and D served as controls. Four days post-transfection,media was removed from transfected cells and assayed for the presence ofHBsAg (see below). Media from untransfected cells was also assayed as abackground control.

Monitoring Cells for Loss of HBV Expression.

Following transfection, cells were monitored for the loss or reductionin HBV expression and replication by measuring HBsAg secretion. Cellswere monitored by assaying the media of transfected cells (and a mediacontrol) at 4 days post-dsRNA administration. The Auszyme ELISA,commercially available from Abbott Labs (Abbott Park, Ill.), was used todetect hepatitis B surface antigen (HBsAg). HBsAg was measured since itis associated not only with viral replication but also with RNApolymerase II initiated transcription of the surface Ag cistron in thetransfected infectious HBV clone. Since HBsAg synthesis can continue inthe absence of HBV replication it is important to down-regulate not onlyviral replication but also replication-independent synthesis of HB sAg.

Results:

Cells transfected with the HBV-specific eiRNA construct exhibited an82-93% decrease in HBsAg at the four-day timepoint relative to thecontrol transfections.

HBV-Specific eiRNA Used in this Experiment

The eiRNA vector encodes a dsRNA mapping to coordinates 2027-2674 of GenBank Accession #AF090840. The sequence therefore includes sequencesderived from both SEQ ID NO:1 and SEQ ID NO:2. More specifically, thesequence includes all of SEQ ID NO:2 and 134 by derived from SEQ IDNO:1.

Example #5 The Down-Regulation of HCV in a Cell Culture Replicon Model

Brief Description

In this experiment, a cell line is created which expresses functionalHCV replicons. Creation of the cell line is as detailed in Lohmann etal. [10]. In this experiment Huh7 cells are used as the parental cellline but in theory any human hepatocyte derived cell line can be used.The cells are then transfected with an HCV specific eiRNA vector. Thepresence of HCV-specific RNA is ascertained by Northern blot analysis asdescribed in Lohmann et al. [10] at days 3-7 post-transfection of eiRNA.

Experimental Protocol: Transfection

Huh7 cells expressing HCV replicons are seeded into six-well plates suchthat they are between 80-90% confluency at the time of transfection. Alltransfections are performed using Lipofectamine™ (InVitrogen) accordingto the manufacturer's directions. In this experiment, cells aretransfected with 1 μg of a T7 RNA polymerase expression plasmid (plasmiddescribed in Example 3) and 1.5 μg of a T7-based eiRNA vector encoding ahairpin RNA comprised of sequences derived from SEQ ID NO:11 (vectordescribed at end of example). Control cells are transfected with 1 μg T7RNA polymerase expression plasmid and 1.5 μg of the HBV-specific (SEQ IDNO:1 specific) T7-based eiRNA vector described in Example 3.Untransfected replicon-expressing HuH 7 cells are included as a secondcontrol. Each transfection mix is made such that ten transfections canbe performed/mix resulting in a total of 20 transfections (10 per mix).At days 3, 4, 5, 6, and 7, two wells of cells/each transfection arelysed and RNA is extracted using standard techniques. Samples areanalyzed simultaneously by Northern blot analysis for the presence ofHCV-specific RNA as described in Lohmann et al. [10].

Results

Cells transfected with the HCV-specific eiRNA vector will show decreasedHCV-specific RNA levels relative to the control cells at everytime-point analyzed.

HCV-Specific eiRNA Vector.

The eiRNA vector is T7-based and encodes a hairpin RNA. One side of thehairpin comprises SEQ ID NO:48.

This sequence is followed by a loop structure of 9 Ts. The second sideof the hairpin contains a sequence that is complementary to the firstside of the hairpin. One skilled in the art can easily design andconstruct hairpin constructs. Note: it is anticipated that other typesof eiRNA vectors driven by other promoters and encoding other types ofRNA structures will have similar effects.

Example #6 Treatment of an HBV/HCV Co-Infection

Brief Description

In this example, cells that are replicating both HBV and HCV repliconsare transfected with an eiRNA vector that encodes both HBV andHCV-specific eiRNA.

Experimental Protocol

Creation of Cell Lines that Contain Both HBV and HCV Replicons.

HuH 7 cells are first engineered to express functional HCV replicons.Creation of the cell line is as detailed in Lohmann et al. [10]. Aftercell line establishment, the cells are transfected with an infectiousHBV replicon plasmid as described in Example 3 and below in the“Transfection of cells” section. In this example, the replicon isderived from the virus sequence found in Gen Bank Accession #s V01460and J02203. Theoretically, it is also possible to first create a cellline that stably expresses the HBV replicon and then use this cell lineto create one that also expresses HCV replicons. It is also possible totransfect the cells simultaneously with both the HBV and HCV repliconsand select and expand cells that are replicating both HBV and HCVreplicons.

Transfection of Cells.

In this example, the HBV and HCV eiRNAs are encoded by separate cistronswithin the same vector. However, similar results are expected if theeiRNAs are encoded within the same cistron or provided by separatevectors. In this example, transcription from each cistron is driven bythe T7 RNA polymerase promoter and T7 RNA polymerase. Each promoter isfollowed by a hairpin eiRNA which in turn is followed by a T7 terminator(FIG. 1). The cistrons in this example are converging but one could alsouse diverging cistrons. It should also be noted that one could use otherexpression systems (including viral) to produce these RNAs and one couldalso use other promoters, e.g., as described elsewhere herein, to driveexpression of these RNAs without significantly affecting efficacy.Selection of the appropriate expression systems and promoters is withinthe skill in this art. Also one could express other eiRNA structures,e.g., as described elsewhere herein, as well as others, described in theliterature in this area. In this example, the HBV eiRNA vector encodessequences derived from SEQ ID NO:1 and the HCV eiRNA vector encodessequences derived from SEQ ID NO:11. Description of vector inserts islocated at the end of this example.

Huh7 cells are seeded into six-well plates such that they are between80-90% confluency at the time of transfection. All transfections areperformed using Lipofectamine™ (Invitrogen) according to themanufacturer's directions. In this experiment, cells are transfectedwith 50 ng of the infectious HBV plasmid, 1 μg of a T7 RNA polymeraseexpression plasmid (description of plasmid is in Example 3), 600 ng ofan eiRNA vector encoding a hairpin RNA comprised of sequences derivedfrom SEQ ID NO:1 (described below and in Example 3), and 600 ng of aneiRNA vector encoding a hairpin RNA comprised of sequences derived fromSEQ ID NO:11 (described below). Control cells are transfected with 50 ngof the HBV plasmid and 1 μg of the T7 RNA polymerase expression plasmid.An inert filler DNA, pGL3-basic (Promega, Madison Wis.), is added to alltransfections where needed to bring total DNA/transfection up to 2.5 μgDNA. Each transfection mix is made such that ten transfections can beperformed/mix resulting in a total of 20 transfections (10 per mix).

Analyses.

Following transfection, cells are monitored for the loss or reduction inHBV expression and replication by measuring HBsAg secretion andDNA-containing viral particle secretion. Cells are monitored by assayingthe media of transfected cells beginning at 2 days post dsRNAadministration and every other day thereafter for a period of threeweeks. The Auszyme ELISA, commercially available from Abbott Labs(Abbott Park, Ill.), is used to detect hepatitis B surface antigen(HBsAg). HBsAg is measured since it is associated not only with viralreplication but also with RNA polymerase II initiated transcription ofthe surface Ag cistron in the transfected infectious HBV clone. SinceHBsAg synthesis can continue in the absence of HBV replication it isimportant to down-regulate not only viral replication but alsoreplication-independent synthesis of HBsAg. Secretion of virionparticles containing encapsidated HBV genomic DNA is also measured. Lossof virion particles containing encapsidated DNA is indicative of a lossof HBV replication. Analysis of virion secretion involves a techniquethat discriminates between naked, immature core particles and envelopedinfectious HBV virions [6]. Briefly, pelleted viral particles from themedia of cultured cells are subjected to Proteinase K digestion todegrade the core proteins. Following inactivation of Proteinase K, thesample is incubated with RQ1 DNase (Promega, Madison, Wis.) to degradethe DNA liberated from core particles. The sample is digested again withProteinase K in the presence of SDS to inactivate the DNase as well asto disrupt and degrade the infectious enveloped virion particle. DNA isthen purified by phenol/chloroform extraction and precipitated. HBVspecific DNA is detected by gel electrophoresis followed by SouthernBlot analysis.

At days 3, 4, 5, 6 and 7, two wells of cells/each transfection(experimental and control) are lysed and RNA is extracted using standardtechniques. Samples are also analyzed by Northern blot analysis for thepresence of HCV-specific RNA as described in Lohmann et al. [10].

Results.

Cells transfected with the HBV-HCV-specific eiRNA vector will showdecreased HCV-specific RNA levels relative to the control cells at everytime-point analyzed. In addition, the levels of HBsAg and HBV viralparticles will also decrease relative to the control transfections.

HCV-Specific eiRNA Sequence.

The eiRNA vector is T7-based and encodes a hairpin RNA. One side of thehairpin comprises SEQ ID NO:48.

This sequence is followed by a loop structure of 9 Ts. The second sideof the hairpin contains a sequence that is complementary to the firstside of the hairpin. One skilled in the art can easily design andconstruct hairpin constructs. Note: it is anticipated that other typesof eiRNA vectors driven by other promoters, including RNA polymerase IIIpromoters, and encoding other types of RNA structures, including varioushairpin structures will have similar effects. Especially desirable are,e.g., “forced” hairpin constructs, partial hairpins capable of beingextended by RNA-dependent RNA polymerase to form dsRNA hairpins, astaught in U.S. Ser. No. 60/399,998P, filed 31 Jul. 2002 andPCT/US2003/024028, filed 31 Jul. 2003, as well as the “udderly”structured hairpins (e.g., multi-hairpin long dsRNA vectors andmulti-short hairpin structures), hairpins with mismatched regions, andmultiepitope constructs as taught in U.S. Ser. No. 60/419,532, filed 18Oct. 2002, and PCT/US2003/033466, filed 20 Oct. 2003, as well as avariety of other dsRNA structures known to those of skill in the art.

HBV-Specific eiRNA-SEQ ID NO:1

The vector is T7-based as described above. The insert encodes aunimolecular hairpin comprised of sequences mapping from coordinate3004-2950 (About 55 bp) of GenBank accession #s V01460 and J02203. Oneregion of the hairpin encodes the sense version of the sequences and thesecond region of the hairpin encodes the antisense version of thissequence. Hairpins can easily be designed and made by those skilled inthe art.

Example #7 Silencing HBV Replication and Expression in a ReplicationCompetent Cell Culture Model (See Example 1) Using Combinations ofHBV-Specific eiRNAs in Multiple Promoter Vectors

As disclosed in PCT/US05/29976, filed 23 Aug. 2005 and in U.S.Provisional Applications entitled “Multiple RNA Pol III PromoterExpression Constructs” (Ser. No. 60/603,622, filed Aug. 23, 2004, andSer. No. 60/629,942, filed Nov. 22, 2004) the teaching of which ishereby incorporated by reference, two or more (preferably 3, 4, 5, 6 ormore) of the shRNA sequences shown in Table I and SEQ ID NO:49 may beencoded in the same plasmid vector in separate cistrons under thecontrol of separate promoters for each shRNA. SEQ ID NO:49 is:

GCCTCGCAGACGAAGGTCTCAAGAGAACTTTGAGACCTTCGTCTGCGAGG C

SEQ ID NO:49 represents the coding strand of a DNA sequence whichencodes an shRNA molecule that targets an HBV conserved region. Thefirst 21 bases of the sequence above are identical to the sense sequenceof HBV mRNA from position 799 to 779 in the HBV genome, strain AYW(numbered according to the complement strand given in Genbank AccessionNo. V01460). This sequence is followed by 9 bases (i.e., AGAGAACTT)representing the loop portion of the shRNA, followed by 21 bases of thereverse complementary sequence to the first 21 bases. (It will beunderstood that the loop sequence serves only to join the complementarysequences which form the double-stranded “stem” and thereforeconsiderable variation in length and nucleotide sequence is acceptablewithin the loop region.) In a preferred embodiment, this DNA sequencewill be placed in an appropriate expression vector operably under thecontrol of a promoter, preferably an RNA polymerase III promoter such7SK, U6, etc. The resulting RNA transcript:

GCCUCGCAGACGAAGGUCUCAAGAGAACUUUGAGACCUUCGUCUGCGAGG Cwill assume a hairpin or stem-loop structure having 21 basepairs in adouble-stranded conformation.

Using methods commonly employed by one skilled in the art of molecularbiology, a single vector encoding two or more, preferably three or more,more preferably four or more, five or more, or all of SEQ ID NO:49, SEQID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23 isconstructed. A particularly preferred embodiment for pharmaceuticalapplications of dsRNA-mediated silencing of the HBV target comprises asingle expression construct encoding under the control of separate RNApolymerase III promoters, shRNAs corresponding to at least SEQ ID NO:19,SEQ ID NO:23, and SEQ ID NO:18, and optionally, SEQ ID NO:49 and/or SEQID NO:21. Such shRNA-expression vectors may advantageously utilize oneor more RNA polymerase III promoters, including U6, 7SK, and H1promoters in several alternative orientations and combinations.Particularly preferred constructs will utilize one or more of the 7SKpromoters as taught in U.S. Provisional Ser. Nos. 60/603,622 and60/629,942. The instant example is thus analogous to Experiment 1 inExample 1 except that instead of introducing one vector with one shRNAat a time, the applicants deliver a single plasmid construct whichexpresses multiple shRNAs.

The advantages of this approach for therapeutic applications of dsRNAsilencing are principally in the economy, simplicity and coordinateddelivery of a single drug entity which comprises multiple differentshRNAs each targeting a different site of the HBV genome. The ability tosimultaneously target multiple sites of a viral genome is highlyadvantageous in preventing the clinically widespread phenomenon of drugresistance (by viral mutation), and the ability to combine dsRNA drugentities against these different target sites in a single delivery agent(the plasmid vectors of this invention) makes this conceptual approachuniquely feasible. While shRNAs, e.g., RNAs corresponding to SEQ IDNO:49, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, and SEQID NO:23 may be produced, e.g., through chemical synthesis or in vitroexpression, and delivered into an animal cell singly and in combination,there are significant advantages in some applications to express withinthe animal cell multiple shRNAs from a single expression vector. In thisexample, the potency of the multiple shRNA expression vectorssignificantly exceeded that of any one of the single vectors used inExperiment 1, as measured by similar assays.

Example #8 Inhibition of Infectious Virions of HCV by dsRNA EffectorMolecules

As a further example of HCV-targeted dsRNAs, the sequences given inTable 16 represent highly conserved coding region sequitopes from the 5′and 3′ untranslated regions of HCV. Each sequence is written as thecoding strand and is used to specify the design of a short hairpin dsRNAeffector molecule comprising the coding sequence as shown in Table 16connected to its reverse complement by a loop or linker sequence asdescribed elsewhere herein. The sequences shown are predicted to beparticularly efficacious as antiviral therapeutic agents because theywere tested in a newly available in vitro HCV replication system capableof producing whole, infectious virions (disclosed in Wakita T,Pietschmann T, Kato T, Date T, Miyamoto M, Zhao Z, Murthy K, HabermannA, Krausslich H G, Mizokami M, Bartenschlager R, Liang T J., “Productionof infectious hepatitis C virus in tissue culture from a cloned viralgenome”, Nat Med 2005 July; 11 (7):791-6; and in Zhong J, Gastaminza P,Cheng G, Kapadia S, Kato T, Burton D R, Wieland S F, Uprichard S L,Wakita T, Chisari F V., “Robust hepatitis C virus infection in vitro”,Proc Natl Acad Sci USA 2005 Jun. 28; 102(26):9294-9). In this system,all viral proteins and viral nucleic acid sequences are present in acell, as in a natural infection. In less complete replicon systems,dsRNA silencing molecules cannot be as rigorously tested as in the newsystem. It is expected that one or more (2, 3, 4, 5, or more) of the HCVsequitopes and their complements could be utilized as duplex dsRNAeffector molecules, short hairpin dsRNA effector molecules, and/orencoded into dsRNA expression vectors capable of expression in vivo in amammalian cell, including a human cell or organism.

TABLE 16 SEQ ID Seq Name NO Sequence (5′ to 3′) HCV5M-5.1 72AAAGGCCTTGTGGTACTGCCT HCV5M-5.3 73 TTGTGGTACTGCCTGATAGGG HCVXM-13 74TAGCTGTGAAAGGTCCGTGAG HCVXM-34 75 ATCTTAGCCCTAGTCACGGCTAGCTG HCVXM-35 76TAGTCACGGCTAGCTGTGAAAGGTCCG

The sequences in Table 17 represent additional preferred highlyconserved at least 19 contiguous base pair HCV sequences from the 5′ UTRof the virus (SEQ ID NO: 11). To generate the dsRNA effector moleculesof the invention, these sequences are used in conjunction with theirreverse complement and, optionally, a loop or linker sequence joiningthe sequence to its reverse complement, when it is desired to form ahairpin dsRNA effector molecule. One or more double-stranded RNAmolecules comprising said conserved 5′ UTR sequences (from SEQ ID NO:11) may advantageously be used in combination with one or more otherdsRNA effector molecules of the invention, including e.g., one or morehighly conserved sequences from the 3′ UTR (SEQ ID NO:27) and/or one ormore at least 19 contiguous base pair sequences from SEQ ID NO. 12.

TABLE 17 HCV 5′ UTR siRNAs SEQ Sequence ID Name Sequence (5′ to 3′) NOHCV5P-1.1 CCTGTGAGGAACTACTGTCTT  77 HCV5P-1.2 ACGCAGAAAGCGTCTAGCCAT  78HCV5P-1.3 CGTCTAGCCATGGCGTTAGTA  79 HCV5P-1.4 GTCTAGCCATGGCGTTAGTAT  80HCV5P-1.5 CTCCCCTGTGAGGAACTACTGTCTT  81 HCV5P-1.6GAGGAACTACTGTCTTCACGCAGAA  82 HCV5P-1.7 GTGAGGAACTACTGTCTTCACGCAGAA  83HCV5P-2.1 GAGCCATAGTGGTCTGCGGAA  84 HCV5P-2.2 GAACCGGTGAGTACACCGGAA  85HCV5P-2.3 ACCGGTGAGTACACCGGAAT  86 HCV5P-2.4 GGGAGAGCCATAGTGGTCTGCGGAA 87 HCV5P-5.1 GGCCTTGTGGTACTGCCTGAT  88 HCV5P-5.2 GCCTTGTGGTACTGCCTGATA 89 HCV5P-5.3 GTACTGCCTGATAGGGTGCTT  90 HCV5P-5.4AAGGCCTTGTGGTACTGCCTGATAGGG  91 HCV5P-5.5 CGAAAGGCCTTGTGGTACTGCCTGATA 92 HCV5P-5.6 CTTGCGAGTGCCCCGGGAGGTCTCGTA  93 HCV5M-1.1ATCACTCCCCTGTGAGGAACT  94 HCV5M-1.2 TTCACGCAGAAAGCGTCTAGC  95 HCV5M-1.3TAGCCATGGCGTTAGTATGAG  96 HCV5M-1.4 ATCACTCCCCTGTGAGGAACTACTG  97HCV5M-1.5 ATCACTCCCCTGTGAGGAACTACTGTC  98 HCV5M-1.6AACTACTGTCTTCACGCAGAAAGCG  99 HCV5M-1.7 AACTACTGTCTTCACGCAGAAAGCGTC 100HCV5M-2.1 ATAGTGGTCTGCGGAACCGGT 101 HCV5M-2.2 TAGTGGTCTGCGGAACCGGTG 102HCV5M-2.3 AACCGGTGAGTACACCGGAATTGCC 103 HCV5M-5.2 AAGGCCTTGTGGTACTGCCTG104 HCV5M-5.4 TACTGCCTGATAGGGTGCTTG 105 HCV5M-5.5TTGTGGTACTGCCTGATAGGGTGCTTG 106 HCV5M-5.6 TACTGCCTGATAGGGTGCTTGCGAG 107HCV5M-5.7 TAGGGTGCTTGCGAGTGCCCCGGG 108 HCV5M-5.8TTGCGAGTGCCCCGGGAGGTCTCGTAG 109

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The invention claimed is:
 1. A composition for inhibiting the expressionof a polynucleotide sequence of hepatitis B virus in an in vivomammalian cell comprising a double-stranded RNA effector molecule,wherein the double-stranded RNA effector molecule comprises (a) SEQ IDNO: 52 and the reverse complement of SEQ ID NO: 52, wherein U issubstituted for T; or (b) SEQ ID NO: 53 and the reverse complement ofSEQ ID NO: 53, wherein U is substituted for T.
 2. The composition ofclaim 1, wherein the double-stranded effector molecule comprises SEQ IDNO: 52 and the reverse complement of SEQ ID NO: 52, wherein U issubstituted for T.
 3. The composition of claim 1, wherein thedouble-stranded effector molecules comprises SEQ ID NO: 53 and thereverse complement of SEQ ID NO: 53, wherein U is substituted for T. 4.The composition of claim 1, wherein the double-stranded RNA effectormolecule comprises SEQ ID NO: 16, wherein U is substituted for T.
 5. Thecomposition of claim 1, wherein the double-stranded RNA effectormolecule comprises SEQ ID NO: 17, wherein U is substituted for T.
 6. Amethod for inhibiting expression of a polynucleotide sequence ofhepatitis B virus in an in vivo mammalian cell comprising administeringto said cell the double-stranded RNA effector molecule of claim
 1. 7.The method of claim 6, wherein the double-stranded effector moleculecomprises SEQ ID NO: 52 and the reverse complement of SEQ ID NO: 52,wherein U is substituted for T.
 8. The method of claim 6, wherein thedouble stranded effector molecule comprises SEQ ID NO: 53 and thereverse complement of SEQ ID NO: 53, wherein U is substituted for T. 9.The method of claim 6, wherein the double-stranded RNA effector moleculecomprises SEQ ID NO: 16, wherein U is substituted for T.
 10. The methodof claim 6, wherein the double-stranded RNA effector molecule comprisesSEQ ID NO: 17, wherein U is substituted for T.